Anti-CD47 antibodies and methods of use

ABSTRACT

Disclosed herein are anti-CD47 antibody molecules, their manufacture and use in treating disorders associated with CD47 expression, for example, certain hematological cancers and solid tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/271,861, filed Sep. 21, 2016, which claims the benefit of, andpriority to, U.S. provisional patent application Ser. No. 62/221,446,filed Sep. 21, 2015, and U.S. provisional patent application Ser. No.62/371,047 filed Aug. 4, 2016, each of which are hereby incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The invention relates generally to molecular biology, immunology andoncology, and, more particularly, the invention relates to antibodiesthat bind CD47.

BACKGROUND OF THE INVENTION

The transmembrane protein CD47, also known as integrin-associatedprotein (IAP), ovarian cancer antigen OA3, Rh-related antigen and MER6,is an immunoglobulin superfamily member involved in multiple cellularprocesses, including cell migration, adhesion and T cell function. CD47was originally identified as a tumor antigen on human ovarian cancer andwas subsequently shown to be expressed on multiple human tumor types,including both hematologic and solid tumors. The interaction betweenCD47 and signal regulatory protein alpha (SIRPα), an inhibitory proteinexpressed on macrophages, prevents phagocytosis of CD47-expressingcells. CD47 is expressed at low levels on virtually all non-malignantcells, and loss of expression or changes in membrane distribution canserve as markers of aged or damaged cells, particularly on red bloodcells (RBC).

However, high expression of CD47 on cancer cells blocks phagocyticuptake, subsequent antigen cross-presentation and T cell activation,which collectively contribute to tumor immune evasion. Certain humanleukemias upregulate CD47 to evade macrophage killing (U.S. Pat. No.8,562,997). In many hematologic cancers, high CD47 expression isbelieved to be associated with poor clinical outcomes, for example,Non-Hodgkin Lymphoma, Acute Lymphocytic Leukemia, etc. (U.S. Pat. No.9,045,541). Similarly, high CD47 expression has been observed in solidtumors such as small cell lung cancer (see, Weiskopf et al. (2016) J.CLIN. INVESTIGATION 126(7): 2610-2620). Agents that block the CD47-SIRPαinteraction can restore phagocytic uptake of CD47⁺ target cells andlower the threshold for macrophage activation, which can enhance theefficacy of therapeutic antibodies with ADCC-enabling activity.

Despite the advances made to date, there is still ongoing need foradditional agents that block the CD47-SIRPα interaction for use in thetreatment of various diseases, including cancers, that are associatedwith elevated levels of CD47 expression.

SUMMARY OF THE INVENTION

Many CD47 antibodies developed to date have been reported to causeaggregation of cells, for example, hemagglutination of humanerythrocytes (see, U.S. Pat. No. 9,045,541). As a consequence, theaggregation of cells, for example, erythrocytes, can limit thetherapeutic utility of anti-CD47 antibodies that have this feature. Theinvention provides antibodies that bind CD47 and disrupt the interactionbetween CD47 and SIRPα, but have little or no hemagglutination activityat the dosage at which the antibody is administered to a subject in needof therapy with an anti-CD47 antibody, for example, a subject with acancer, for example, a hematologic cancer or solid tumor.

The invention is based, in part, on the development and characterizationof a series of antibody molecules that bind with high affinity to humanCD47, block the CD47-SIRPα interaction, and promote macrophage-mediatedphagocytic clearance of CD47-expressing cancer cells while inducinglittle or no hemagglutination of red blood cells. The anti-CD47 antibodymolecules disclosed herein display significant tumor growth inhibitionin models for multiple myeloma, diffuse large B cell lymphoma (DLBCL),and Burkitt's lymphoma, as a single agent and in combination with anopsonizing antibody. The anti-CD47 antibody molecules disclosed hereincan be used (alone or in combination with other agents or therapeuticmodalities) to treat, prevent and/or diagnose disorders such as cancerand precancerous conditions. The CD47 antibodies described herein areuseful in treating, delaying the progression of, preventing relapse of,or alleviating one or more symptoms of a cancer or a precancerouslesion, and are useful in treating hematological malignancies and/ortumors.

In certain embodiments, the anti-CD47 antibody molecules describedherein are capable of blocking the interaction between CD47 and itscognate SIRPα ligand, without causing significant, or detectable,hemagglutination of erythrocytes, e.g., human erythrocytes. For example,the antibody molecules cause less hemagglutination of human erythrocytesthan a reference anti-CD47 antibody, or cause less than 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, or 10% or less hemagglutination of humanerythrocytes relative to a reference anti-CD47 antibody. Exemplaryreference antibodies include B6H12, MABL, BRIC126, and CC2C6.

In one embodiment, the anti-CD47 antibody molecules described hereincause a potent blocking of the interaction between CD47 and SIRPαwithout causing a significant level of hemagglutination of erythrocytes,as well as potent anti-cancer activity. For example, the anti-CD47antibody molecules described block at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 99% of theinteraction between CD47 and SIRPα as compared to the level ofinteraction between CD47 and SIRPα in the absence of the anti-CD47antibody molecules described herein. Optionally, the antibody moleculesalso cause less hemagglutination of human erythrocytes than a referenceanti-CD47 antibody, or cause less than 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, or 10% or less hemagglutination of human erythrocytes relativeto a reference anti-CD47 antibody. Exemplary reference antibodiesinclude B6H12, MABL, BRIC126, and CC2C6.

In one embodiment, the anti-CD47 antibody molecules described herein donot phagocytose red blood cells to a significant or detectable level. Inanother embodiment, the anti-CD47 antibody molecules have reduced (e.g.,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) phagocyticactivity towards red blood cells relative to a reference anti-CD47antibody, e.g., as determined by a phagocytosis assay described herein.Exemplary reference antibodies include B6H12, MABL, BRIC126, and CC2C6.

In another embodiment, the anti-CD47 antibody molecules described hereinenhance macrophage activity. For example, the antibody molecules enhancethe phagocytic activity of a macrophage, e.g., an unpolarizedmacrophage, or an M1 or M2 polarized macrophage. In one embodiment, thephagocytic activity is enhanced, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90%, relative to a macrophage in the absence of ananti-CD47 antibody molecule described herein.

In one embodiment, the anti-CD47 antibody molecules described hereinenhance macrophage phagocytic activity towards a cancer cell, e.g., anAML cell. In one embodiment, the phagocytic activity is enhanced, e.g.,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, relative to amacrophage in the absence of an anti-CD47 antibody molecule describedherein.

In one embodiment, the anti-CD47 antibody molecules described herein,when used in combination with an opsonizing antibody (e.g., one or moreof, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD38 antibody,or an anti-HER2/neu receptor antibody) enhance the anti-tumor effect ofthe combination, relative to the anti-tumor effect of each antibodyindividually. In another embodiment, the anti-tumor effect of thecombination is enhanced, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90% or higher, relative to the activity of either the anti-CD47antibody molecule or the opsonizing antibody individually.

In one aspect, the anti-CD47 antibody molecule comprises: a heavy chaincomplementarity determining region 1 (HC CDR1) of the amino acidsequence set forth in SEQ ID NO: 7, a heavy chain complementaritydetermining region 2 (HC CDR2) of the amino acid sequence set forth inSEQ ID NO: 8, a heavy chain complementarity determining region 3 (HCCDR3) of the amino acid sequence set forth in SEQ ID NO: 9; and a lightchain complementarity determining region 1 (LC CDR1) of the amino acidsequence set forth in SEQ ID NO: 10, a light chain complementaritydetermining region 2 (LC CDR2) of the amino acid sequence set forth inSEQ ID NO: 11, and a light chain complementarity determining region 3(LC CDR3) of the amino acid sequence set forth in SEQ ID NO: 12.

In an embodiment, an antibody molecule of the invention comprises one orboth of (a) and (b), wherein (a) and (b) are as follows:

(a)(i) light chain CDR1, CDR2 and CDR3, e.g., Chothia or Kabat lightchain CDRs, from SEQ ID NO: 16,

(a)(ii) light chain CDR1 of SEQ ID NO: 10, light chain CDR2 of SEQ IDNO: 11, and light chain CDR3 of SEQ ID NO: 12,

(a)(iii) light chain CDRs CDR1, CDR2 and CDR3, that collectively, differby no more than 1, 2, 3, 4, 5, or 6 amino acid residues from the lightchain CDRs of (a)(i) and (a)(ii);

(a)(iv) a light chain variable region of SEQ ID NO: 6;

(a)(v) an antigen binding fragment of SEQ ID NO: 6;

(a)(vi) an amino acid sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, residues from the sequence of (a)(iv) or a)(v);

(a)(vii) an amino acid sequence that is substantially identical (e.g.,at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical) to the sequenceof (a)(iv) or (a)(v); and

(b)(i) heavy chain CDR1, CDR2 and CDR3, e.g., Chothia or Kabat heavychain CDRs, from SEQ ID NO: 15,

(b)(ii) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO:8, and heavy chain CDR3 of SEQ ID NO: 9,

(b)(iii) heavy chain CDRs CDR1, CDR2 and CDR3, that collectively, differby no more than 1, 2, 3, 4, 5, or 6 amino acid residues from the heavychain CDRs of (b)(i) and (b)(ii);

(b)(iv) a heavy chain variable region of SEQ ID NO: 4;

(b)(v) an antigen binding fragment of SEQ ID NO: 4;

(b)(vi) an amino acid sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, residues from the sequence of (b)(iv) or (b)(v);and

(b)(vii) an amino acid sequence that is substantially identical (e.g.,at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical) to the sequenceof (b)(iv) or (b)(v).

In an embodiment, the antibody molecule comprises (a)(i) and (b)(i).

In an embodiment, the antibody molecule comprises (a)(ii) and (b)(ii).

In an embodiment, the antibody molecule comprises (a)(iii) and (b)(iii).

In an embodiment, the antibody molecule comprises (a)(iv) and (b)(iv).

In an embodiment, the antibody molecule comprises (a)(v) and (b)(v).

In an embodiment, the antibody molecule comprises (a)(vi) and (b)(vi).

In an embodiment, the antibody molecule competes for binding to CD47with an antibody described herein, e.g., competes for binding with theantibody 2.3D11, discussed hereinbelow.

In an embodiment, the antibody molecule described herein binds the sameor an overlapping epitope on CD47 as an antibody described herein, e.g.,the antibody 2.3D11.

In an embodiment, the anti-CD47 antibody molecule is a bispecificantibody molecule. For example, the bispecific antibody molecule cancomprise a first binding specificity to CD47, e.g., an antibody thatbinds CD47 as described herein, and a second binding specificity. Thesecond binding specificity can be imparted via a binding domain obtainedfrom an opsonizing antibody, e.g., an antibody that binds CD19, CD20,CD38, or HER2/neu receptor.

It is understood that the variable region sequences of the antibodiesdescribed herein can be linked to a variety of constant regionsequences. For example, in one embodiment, the anti-CD47 antibodymolecule can have a wild-type heavy chain constant region (Fc). Inanother embodiment, the anti-CD47 antibody molecule can have a mutatedform of a heavy chain constant region. In one embodiment, the heavychain constant region is chosen from, e.g., a heavy chain constantregion of an IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;preferably, chosen from, e.g., a heavy chain constant region of IgG1,IgG2, IgG3, and IgG4. In one embodiment, the anti-CD47 antibody moleculehas an IgG1 heavy chain constant region, e.g., a wild-type or mutantIgG1 heavy chain constant region. In another embodiment, the anti-CD47antibody molecule has an IgG4 heavy chain constant region, e.g., awild-type or mutant IgG4 heavy chain constant region. In one embodiment,the IgG4 heavy chain constant region comprises one or both of thesubstitutions, serine to proline at position 228 (S228P) and leucine toglutamate at position 235 (L235E), e.g., according to EU numbering.

In another embodiment, the anti-CD47 antibody molecule has a light chainconstant region chosen from, e.g., the light chain constant regions ofkappa or lambda.

In another aspect, the invention also provides compositions comprisingan anti-CD47 antibody molecule described herein and at least onepharmaceutically acceptable carrier or diluent. For example, thecomposition comprises an isolated anti-CD47 antibody molecule,comprising: a heavy chain complementarity determining region 1 (HC CDR1)of the amino acid sequence set forth in SEQ ID NO: 7, a heavy chaincomplementarity determining region 2 (HC CDR2) of the amino acidsequence set forth in SEQ ID NO: 8, a heavy chain complementaritydetermining region 3 (HC CDR3) of the amino acid sequence set forth inSEQ ID NO: 9; and a light chain complementarity determining region 1 (LCCDR1) of the amino acid sequence set forth in SEQ ID NO: 10, a lightchain complementarity determining region 2 (LC CDR2) of the amino acidsequence set forth in SEQ ID NO: 11, and a light chain complementaritydetermining region 3 (LC CDR3) of the amino acid sequence set forth inSEQ ID NO: 12, and at least one pharmaceutically acceptable carrier ordiluent.

In one embodiment, the isolated anti-CD47 antibody molecules disclosedherein, comprise a heavy chain variable region (VH) of the amino acidsequence set forth in SEQ ID NO: 4, or a sequence substantiallyidentical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or 99%identical) to SEQ ID NO: 4; and a light chain variable region (VL) ofthe amino acid sequence set forth in SEQ ID NO: 6, or a sequencesubstantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or99% identical) to SEQ ID NO: 6.

In one embodiment, the composition comprises an isolated anti-CD47antibody molecule having a heavy chain variable region (VH) of the aminoacid sequence set forth in SEQ ID NO: 4, or a sequence substantiallyidentical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or 99%identical) to SEQ ID NO: 4; and a light chain variable region (VL) ofthe amino acid sequence set forth in SEQ ID NO: 6, or a sequencesubstantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or99% identical) to SEQ ID NO: 6, and at least one pharmaceuticallyacceptable carrier or diluent.

In one embodiment, the isolated anti-CD47 antibody molecule comprises: aheavy chain of the amino acid sequence set forth in SEQ ID NO: 15, or asequence substantially identical (e.g., at least 85%, 90%, 92%, 95%,97%, 98%, or 99% identical) to SEQ ID NO: 15; and a light chain of theamino acid sequence set forth in SEQ ID NO: 16, or a sequencesubstantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or99% identical) to SEQ ID NO: 16.

In one embodiment, the composition comprises an isolated anti-CD47antibody molecule comprising: a heavy chain of the amino acid sequenceset forth in SEQ ID NO: 15, or a sequence substantially identical (e.g.,at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to SEQ ID NO:15; and a light chain of the amino acid sequence set forth in SEQ ID NO:16, or a sequence substantially identical (e.g., at least 85%, 90%, 92%,95%, 97%, 98%, or 99% identical) to SEQ ID NO: 16, and at least onepharmaceutically acceptable carrier or diluent.

In embodiments of any of the aforementioned antibody molecules orcompositions, an anti-CD47 antibody molecule comprising a substantiallyidentical heavy chain and/or light chain sequence to a reference SEQ IDNO (e.g., the heavy chain of SEQ ID NO: 15 or the light chain of SEQ IDNO: 16) comprises one, two, or three VH CDRs, and/or one, two, or threeVL CDRs, having an amino acid sequence that is identical to thecorresponding reference CDR sequence.

In another aspect, the invention provides a method of treating (orpreventing) cancer in a subject, the method comprising administering ananti-CD47 antibody molecule or a composition comprising an isolatedanti-CD47 antibody molecule to the subject. For example, the inventionprovides a method of treating (or preventing) cancer in a subject, themethod comprising: administering an anti-CD47 antibody moleculedescribed herein, or a composition comprising an isolated anti-CD47antibody molecule described herein, to the subject.

In one embodiment, the anti-CD47 antibody molecule comprises: a heavychain complementarity determining region 1 (HC CDR1) of the amino acidsequence set forth in SEQ ID NO: 7, a heavy chain complementaritydetermining region 2 (HC CDR2) of the amino acid sequence set forth inSEQ ID NO: 8, a heavy chain complementarity determining region 3 (HCCDR3) of the amino acid sequence set forth in SEQ ID NO: 9; and a lightchain complementarity determining region 1 (LC CDR1) of the amino acidsequence set forth in SEQ ID NO: 10, a light chain complementaritydetermining region 2 (LC CDR2) of the amino acid sequence set forth inSEQ ID NO: 11, and a light chain complementarity determining region 3(LC CDR3) of the amino acid sequence set forth in SEQ ID NO: 12.

In certain embodiments, a composition, e.g., a composition comprising ananti-CD47 antibody described herein, is administered by a mode selectedfrom the group consisting of: parenteral, subcutaneous, intramuscular,intravenous, intra-articular, intra-abdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intraspinal, intrasynovial, rectal, buccal,sublingual, intranasal, and transdermal delivery. In certainembodiments, the composition is administered subcutaneously. In certainembodiments, the composition is administered intravenously.

In certain embodiments, an anti-CD47 antibody molecule, for example, ananti-CD47 antibody described herein or a composition comprising ananti-CD47 antibody molecule or a composition comprising an anti-CD47antibody described herein, is administered in combination with achemotherapeutic agent or a second therapeutic antibody molecule. Forexample, in one embodiment, an anti-CD47 antibody molecule orcomposition, e.g., an anti-CD47 antibody molecule or compositiondescribed herein, is administered in combination with an opsonizingantibody. Without wishing to be bound by theory, the opsonizing antibodycan facilitate phagocytosis or antibody dependent cellular cytotoxicity(ADCC), or both, of a target cell, e.g., a cancer cell. In oneembodiment, the antigen binding portion of the opsonizing antibody bindsto a target antigen, whereas the Fc portion of the opsonizing antibodybinds to an Fc receptor on a phagocyte. In other embodiments, theantigen binding portion of the opsonizing antibody binds to a targetantigen, whereas the Fc portion of the opsonizing antibody binds to animmune effector cell, e.g., via its Fc domain, thus triggering targetcell lysis by the bound effector cell (e.g., monocytes, neutrophils andnatural killer cells).

In certain embodiments, the opsonizing antibody is an anti-CD20 antibodymolecule, for example, rituximab. In certain embodiments, the opsonizingantibody is an anti-CD19 antibody molecule. In certain embodiments, theopsonizing antibody is an anti-CD38 antibody molecule. In certainembodiments, the opsonizing antibody is an anti-HER2/neu receptorantibody molecule.

In certain embodiments, the antibody molecules can be used to treat ahematological cancer, for example, a hematological cancer selected fromthe group consisting of: acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), Non-Hodgkin lymphoma (e.g., diffuse large Bcell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, Blymphoblastic leukemia/lymphoma, and Burkitt's lymphoma),B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocyticleukemia/small lymphocytic lymphoma, chronic lymphocytic leukemia (CLL),e.g., transformed CLL, Richter's syndrome, chronic myelocytic leukemia(CML), follicular lymphoma, multiple myeloma, myelofibrosis,polycythemia vera, cutaneous T-cell lymphoma, monoclonal gammopathy ofunknown significance (MGUS), myelodysplastic syndrome (MDS),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphomaand anaplastic large cell lymphoma.

In one embodiment, the cancer is a hematological cancer chosen frommultiple myeloma, diffuse large B cell lymphoma, AML, CLL, e.g.,transformed CLL, Richter's syndrome, or follicular lymphoma. In certainembodiments, the antibody molecules can be used to treat a solid tumor.In certain embodiments, the cancer is selected from the group consistingof lung (e.g., non-small cell lung cancer, small cell lung cancer),pancreas, breast, liver, ovary, testicle, kidney, bladder, spine, brain,cervix, endometrial, colon/rectum, anus, endometrium, esophagus,gallbladder, gastrointestinal tract, skin, prostate, pituitary, stomach,uterus, vagina, and thyroid. In certain embodiments, the solid tumor isN-methyl-D-aspartate receptor (NMDA receptor) positive teratoma. Incertain embodiments, the cancer is a cancer associated with ascitesselected from breast cancer, colon cancer, stomach cancer, pancreaticcancer, uterine cancer, and ovarian cancer. In one embodiment, thecancer associated with ascites is an adenocarcinoma.

In certain embodiments, the method of preventing a cancer comprisestreating a precancerous condition or a condition associated withincreased risk of developing cancer. Exemplary precancerous conditionsinclude plasma cell dyscrasias, including a monoclonal gammopathy ofunknown significance (MGUS), which are associated with an increased riskfor development of multiple myeloma and other hematologic malignancies.

In another aspect, the invention provides one or more isolated nucleicacid molecules that encode at least a portion (for example, one of theheavy or light chain sequences) of the anti-CD47 antibody moleculesdescribed herein.

In one embodiment, the nucleic acid molecule comprises a nucleic acidsequence encoding a heavy chain complementarity determining region 1 (HCCDR1) of the amino acid sequence set forth in SEQ ID NO: 7, a heavychain complementarity determining region 2 (HC CDR2) of the amino acidsequence set forth in SEQ ID NO: 8, and a heavy chain complementaritydetermining region 3 (HC CDR3) of the amino acid sequence set forth inSEQ ID NO: 9, and/or a light chain complementarity determining region 1(LC CDR1) of the amino acid sequence set forth in SEQ ID NO: 10, a lightchain complementarity determining region 2 (LC CDR2) of the amino acidsequence set forth in SEQ ID NO: 11, and a light chain complementaritydetermining region 3 (LC CDR3) of the amino acid sequence set forth inSEQ ID NO: 12. It is contemplated that the nucleic acid encodes (i) HCCDR1, HC CDR2, and HC CDR3; (ii) LC CDR1, LC CDR2, and LC CDR3; or (iii)HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3.

In certain embodiments, the one or more isolated nucleic acid moleculesencode an anti-CD47 antibody molecule comprising: a heavy chain variableregion (VH) of the amino acid sequence set forth in SEQ ID NO: 4, or asequence substantially identical (e.g., at least 85%, 90%, 92%, 95%,97%, 98%, or 99% identical) to SEQ ID NO: 4; and/or a light chainvariable region (VL) of the amino acid sequence set forth in SEQ ID NO:6, or a sequence substantially identical (e.g., at least 85%, 90%, 92%,95%, 97%, 98%, or 99% identical) to SEQ ID NO: 6.

In certain embodiments, the one or more isolated nucleic acid moleculesencode an anti-CD47 antibody molecule comprising: a heavy chain of theamino acid sequence set forth in SEQ ID NO: 15, or a sequencesubstantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or99% identical) to SEQ ID NO: 15; and/or a light chain of the amino acidsequence set forth in SEQ ID NO: 16, or a sequence substantiallyidentical (e.g., at least 85%, 90%, 92%, 95%, 97%, 98%, or 99%identical) to SEQ ID NO: 16.

In certain embodiments, the one or more isolated nucleic acid moleculesencode an anti-CD47 antibody molecule comprising: a heavy chain variableregion (VH) of the amino acid sequence set forth in SEQ ID NO: 4, or asequence substantially identical (e.g., at least 85%, 90%, 92%, 95%,97%, 98%, or 99% identical) to SEQ ID NO: 4; and/or a light chainvariable region (VL) of the amino acid sequence set forth in SEQ ID NO:6, or a sequence substantially identical (e.g., at least 85%, 90%, 92%,95%, 97%, 98%, or 99% identical) to SEQ ID NO: 6.

In another aspect, the invention provides a vector comprising a nucleicacid molecule described herein (e.g., one or more isolated nucleic acidmolecules encoding an anti-CD47 antibody molecule comprising a nucleicacid sequence encoding a heavy chain complementarity determining region1 (HC CDR1) of the amino acid sequence set forth in SEQ ID NO: 7, aheavy chain complementarity determining region 2 (HC CDR2) of the aminoacid sequence set forth in SEQ ID NO: 8, a heavy chain complementaritydetermining region 3 (HC CDR3) of the amino acid sequence set forth inSEQ ID NO: 9, and/or a light chain complementarity determining region 1(LC CDR1) of the amino acid sequence set forth in SEQ ID NO: 10, a lightchain complementarity determining region 2 (LC CDR2) of the amino acidsequence set forth in SEQ ID NO: 11, and a light chain complementaritydetermining region 3 (LC CDR3) of the amino acid sequence set forth inSEQ ID NO: 12).

In another aspect, the invention provides cells comprising one or morevectors described herein (e.g., vectors comprising a nucleic acidmolecule described herein (e.g., one or more isolated nucleic acidmolecules encoding an anti-CD47 antibody molecule, comprising a nucleicacid sequence encoding a heavy chain complementarity determining region1 (HC CDR1) of the amino acid sequence set forth in SEQ ID NO: 7, aheavy chain complementarity determining region 2 (HC CDR2) of the aminoacid sequence set forth in SEQ ID NO: 8, a heavy chain complementaritydetermining region 3 (HC CDR3) of the amino acid sequence set forth inSEQ ID NO: 9, and/or a light chain complementarity determining region 1(LC CDR1) of the amino acid sequence set forth in SEQ ID NO: 10, a lightchain complementarity determining region 2 (LC CDR2) of the amino acidsequence set forth in SEQ ID NO: 11, and a light chain complementaritydetermining region 3 (LC CDR3) of the amino acid sequence set forth inSEQ ID NO: 12)).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a line graph depicting the binding of a biotinylated fusionprotein of SIRPα (SIRPα-Fc-bio) to Jurkat cells in the presence ofcertain antibodies including the anti-CD47 antibodies 2D3, B6H12,AB6.12-IgG4PE, and 2.3D11, and a hIgG control.

FIG. 2A is a line graph depicting the binding of B6H12-FITC to DU-145cells pre-incubated with increasing concentrations of unlabeledantibodies 2.3D11, B6H12 or isotype control. FIG. 2B is a line graphdepicting the binding of biotinylated 2.3D11 (2.3D11-bio) to DU-145cells pre-incubated with increasing concentrations of unlabeledantibodies 2.3D11, B6H12 or isotype control. Binding of 2.3D11-bio wasdetecting using SA-FITC. FIG. 2C is a graph depicting the binding ofantibody B6H12-FITC (18 μg/ml) to Panc-1 cells, co-incubated with orwithout unlabeled 2.3D11 antibody at 0.67, 2, 6 or 18 μg/ml. Staininglevels are compared to binding of 18 μg/ml of isotype control antibodymouse IgG1-FITC (IC).

FIG. 3A is a graph depicting the binding of the indicated anti-CD47antibodies and mIgG1 control to human red blood cells. FIG. 3B is agraph depicting the binding of the indicated anti-CD47 antibodies andmIgG1 control to cynomolgus (cyno) red blood cells. FIG. 3C is a linegraph depicting the binding of the indicated anti-CD47 antibodies andmIgG1 control to human red blood cells. FIG. 3D is a line graphdepicting the binding of the indicated anti-CD47 antibodies and mIgG1control to cyno red blood cells.

FIG. 4A-B is a fluorescence activated cell sorting (FACS) dot plotdepicting the level of phagocytosed target cells in the presence ofcontrol antibody polyclonal hIgG (FIG. 4A) or the anti-CD47 antibody2.3D11 (FIG. 4B). Events shown are gated on CD14 and doublets areexcluded. FIG. 4C is a legend that identifies the sector correspondingto phagocytosed targets in FIG. 4A and FIG. 4B.

FIG. 5 is a bar chart depicting the percent of macrophages that havephagocytosed target Jurkat cells in the presence of &-control antibodies(monoclonal murine IgG1; mIgG1 or polyclonal human IgG; hIgG) or theindicated anti-CD47 antibody at 1 μg/ml (open bars) or 10 μg/ml (hatchedbars).

FIG. 6 is a bar chart depicting the percent of macrophages that havephagocytosed Raji target cells in the presence of control antibody(hIgG), B6H12, or 2.3D11 and either a control human IgG antibody or theanti-CD20 antibody rituximab. The anti-CD47 antibodies (B6H12, and2.3D11) and the anti-CD20 antibodies were used at sub-optimalconcentrations (0.3 μg/ml and 0.1 μg/ml, respectively) in order toobserve cooperative effects. Isotype control antibody was used atmatching concentrations.

FIG. 7A is a bar chart depicting the percent of macrophages that havephagocytosed target Raji cells in the presence of control antibody orthe indicated anti-CD47 antibody. FIG. 7B is a bar chart depicting thepercent of macrophages that have phagocytosed Jurkat target cells in thepresence of control antibody or the indicated anti-CD47 antibody. FIG.7C is a line graph depicting the level of CD47 expression, as determinedby 2.3D11 staining, on Raji, Jurkat, and DU-145 cells. Cells wereincubated with the indicated concentrations of 2.3D11-bio and stainingwas detected with SA-FITC.

FIG. 8 is a photograph of a 96 well plate depicting the hemagglutinationof human red blood cells in the presence of a dose curve for eachindicated anti-CD47 antibody or control.

FIGS. 9A and 9B are bar charts depicting the percent of macrophages thathave phagocytosed human red blood cells (FIG. 9A) and cyno red bloodcells (FIG. 9B) in the presence of each of the indicated anti-CD47antibodies or control.

FIGS. 10A-C are line graphs summarizing the effects of the anti-CD47antibodies 2.3D11 IgG4 or 2.3D11 IgG4mt, alone or in combination withrituximab, in the Raji lymphoma xenograft model. FIG. 10A shows theanti-tumor effects of the anti-CD47 antibodies in the Raji lymphomaxenograft model. Isotype control (filled circles) 2.3D11 IgG4mt (opendiamonds) and 2.3D11 IgG4 (filled triangles) were dosed at 200 μg permouse, t.i.w. for 3 weeks. Tumor volume measurement are presented asmeans+/−SEM (n=10). FIG. 10B shows the anti-tumor effects of 2.3D11IgG4mt in combination with rituximab in the Raji lymphoma xenograftmodel. Isotype control (filled circles) and 2.3D11 IgG4mt (opendiamonds) were dosed at 200 μg t.i.w., rituximab (open circles) wasdosed at 5 mg/kg q.w. and the 2.3D11 IgG4mt/rituximab combination (opensquares) was dosed at 200 μg t.i.w. and 5 mg/kg q.w., respectively; allantibodies were dosed for three weeks. Tumor volume measurements arepresented as mean+/−SEM (n=8). FIG. 10C shows the anti-tumor effects of2.3D11 IgG4 in combination with rituximab in the Raji lymphoma xenograftmodel. Isotype control (filled circles) and 2.3D11 IgG4 (filledtriangles) were dosed at 100 μg t.i.w., rituximab (open circles) wasdosed at 5 mg/kg q.w. and the 2.3D11 IgG4/rituximab combination (opensquares) was dosed at 100 μg t.i.w. and 5 mg/kg q.w., respectively; allantibodies were dosed for three weeks. Tumor volume measurements arepresented as mean+/−SEM (n=8).

FIG. 11. is a line graph showing the percent of CD14+ cells that wereCFSE+ in a red blood cell phagocytosis assay. Human red blood cells wereisolated from healthy donors and labeled with CFSE. RBC were culturedwith day 7 human macrophages in the phagocytosis assay described inExample 4 at a target-to-effector ratio of 10:1. Representative datafrom one of three donors shown; filled squares indicates 2.3D11 IgG1,filled circles indicate 2.3D11 IgG4, filled triangles indicate 2.3D11IgG4mt, open circles indicate human IgG4 isotype control, open trianglesindicate murine IgG1 isotype control, and closed diamonds indicateB6H12.

FIGS. 12A-12D are bar graphs showing the percent of CD14+ cells thatwere CFSE+ in a phagocytosis assay with polarized macrophages. Primaryhuman monocytes were differentiated in media containing 100 ng/mLrecombinant human macrophage colony-stimulating factor (M-CSF) for 6days. On the sixth day, macrophages were replated in the presence ofeither M-CSF alone (FIG. 12A), M-CSF plus interleukin-10 (IL-10),transforming growth factor β (TGFβ) and interleukin-4 (IL-4) (FIG. 12B),M-CSF plus interferon γ and lipopolysaccharide (LPS) (FIG. 12C), orM-CSF plus Dexamethasone (FIG. 12D) overnight. Phagocytosis assays wereperformed on day 7, as described in Example 4, using CFSE-labeled Jurkatcells as targets. Antibody concentrations used are indicated by: openbars, 0.08 μg/mL; lined bars, 0.4 μg/mL; hatched bars, 2 μg/mL.

FIG. 13 is a chart showing the percent of CD14+ cells that were CFSE+ ina phagocytosis assay of tumor cells. Primary human monocytes weredifferentiated in media containing 100 ng/mL recombinant humanmacrophage colony-stimulating factor (M-CSF) for 7 days. Frozen bonemarrow samples from AML patients were thawed, labelled with CFSE andcultured with differentiated macrophages for 2 hours at atarget-to-effector ratio of 1:1, in the presence of the indicatedantibodies. Phagocytosis was quantitated as described in Example 4.Results from three independent experiments are pooled. Each datapointshown is an individual donor. * p≦0.05; ** p≦0.01 as measured by anupaired Student's t-Test.

FIG. 14 is a graph showing tumor volume in the Raji lymphoma xenograftmodel after treatment with the anti-CD47 antibodies 2.3D11 IgG1, 2.3D11IgG4, or 2.3D11 IgG4mt. SCID-Beige mice were implanted subcutaneouslywith Raji tumor cells and randomized when the tumors reached ˜100 mm³ toreceive the indicated antibodies at 200 μg/mouse t.i.w. for 3 weeks.Closed circles indicate human polyclonal IgG, open circles indicate2.3D11 IgG4mt, open diamonds indicate 2.3D11 IgG4, and open squaresindicate 2.3D11 IgG1. In the 2.3D11 IgG4mt arm, 2 tumors reached 2000mm³ at day 38 and the mice were terminated, so average tumor volumeswere not reported after this timepoint.

FIG. 15 is a bar chart showing the percent of CD14+ cells that wereCFSE+ in a phagocytosis assay of multiple myeloma cells. A primarymultiple myeloma bone marrow sample was CFSE labelled and co-culturedwith differentiated human macrophages at a ratio of 2:1, in the presenceof 10 μg/mL isotype control, 10 μg/mL of 2.3D11, anti-human CD38-hIgG1,or both. Note that single-agent conditions were supplemented with 10μg/mL of isotype control.

FIG. 16 is a graph showing the decrease in tumor volume in mice treatedwith 2.3D11 IgG4 either alone or in combination with Daratumumab. CB.17SCID mice were implanted with H929 tumor cells. When tumors reached anaverage size of 100-150 mm³, animals were randomized to control ortreatment arms. Filled circles indicate isotype control, filled squaresindicate Daratumumab administered as a single dose at 10 μg/mouse,filled triangles indicate 2.3D11 IgG4 administered three times per weekfor 3 weeks at 30 μg/mouse, and open diamonds indicate a combination of2.3D11 IgG4 and Daratumumab at the monotherapy doses.

FIG. 17 is a graph showing the percent of CD14+ cells that were CFSE+ ina phagocytosis assay of chronic lymphocytic leukemia (CLL) cells.CD19⁺/CD5⁺ tumor cells from the peripheral blood of a CLL patient wereCFSE labelled and co-cultured with differentiated human macrophages at aratio of 2:1, in the presence of different concentrations of 2.3D11 IgG4(circles) and isotype control (triangles).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to antibody molecules that specifically bind CD47,including human CD47, and modulate, e.g., block, inhibit, reduce,antagonize, neutralize or otherwise interfere with the interactionbetween CD47 and signal regulatory protein α (SIRPα), without causingsignificant aggregation of cells, for example, hemagglutination oferythrocytes. Many other CD47 antibodies, e.g., B6H12, MABL, BRIC126,and CC2C6, have been reported to cause hemagglutination of humanerythrocytes (e.g., U.S. Pat. No. 9,045,541, Uno S, Kinoshita Y, Azuma Yet al. (2007) ONCOL. REP. 17: 1189-94; Kikuchi Y, Uno S, Yoshimura Y etal. (2004) BIOCHEM. BIOPHYS. RES. COMMUN. 315: 912-8). The aggregationof cells represents an important limitation of many therapeuticanti-CD47 antibodies. The anti-CD47 antibody molecules of the presentdisclosure, including the 2.3D11 antibody molecule, avoid theundesirable effects of agglutination, for example hemagglutination,thereby increasing the efficacy of therapeutically targeting CD47, whilemaintaining the ability to block the interaction of CD47 with SIRPα,thereby promoting phagocytosis of cells expressing CD47. It has alsobeen discovered that the 2.3D11 antibody unexpectedly cross competeswith the anti-CD47 antibody B6H12 for binding to CD47, even though,unlike B6H12, 2.3D11 does not induce hemagglutination or red blood cellphagocytosis.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The techniques and procedures described herein are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See e.g., Sambrooket al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell-known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

CD47

CD47, also known as integrin-associated protein (IAP), ovarian cancerantigen OA3, Rh-related antigen and MER6, is a multi-spanningtransmembrane receptor belonging to the immunoglobulin superfamily CD47expression and/or activity has been implicated in a number of diseasesand disorders, e.g., cancer. CD47 interacts with SIRPα(signal-regulatory-protein α) on macrophages and thereby inhibitsphagocytosis.

An amino acid sequence of an exemplary human CD47 protein is provided inSEQ ID NO: 1 (NCBI Reference Sequence: NP_001768.1). An mRNA sequenceencoding an exemplary human CD47 protein is provided in SEQ ID NO: 2(NCBI Reference Sequence: NM_001777).

Antibody Molecules

As used herein, the term “antibody molecule” refers to a polypeptide orcombination of polypeptides that comprise sufficient sequence from animmunoglobulin heavy chain variable region and/or sufficient sequencefrom an immunoglobulin light chain variable region, to specifically bindto an antigen. The term comprises full length antibodies as well asfragments thereof, e.g., Fab, F(ab′) or F(ab′)₂ fragments. Typically, anantibody molecule comprises heavy chain CDR1, CDR2, and CDR3 and lightchain CDR1, CDR2, and CDR3 sequences. Antibody molecules include human,humanized, CDR-grafted antibodies and antigen binding fragments thereof.In certain embodiments, an antibody molecule comprises a protein thatcomprises at least one immunoglobulin variable region segment, e.g., anamino acid sequence that provides an immunoglobulin variable domain orimmunoglobulin variable domain sequence.

The VH or VL chain of the antibody molecule can further include all orpart of a heavy or light chain constant region, to thereby form a heavyor light immunoglobulin chain, respectively. The antibody molecule canbe a typical tetramer of two heavy immunoglobulin chains and two lightimmunoglobulin chains where the two heavy chains are linked byoptionally at least one disulfide bond and each pair of heavy and lightchains are linked by a disulfide bond.

An antibody molecule can comprise one or both of a heavy (or light)chain immunoglobulin variable region segment. As used herein, the term“heavy (or light) chain immunoglobulin variable region segment,” refersto an entire heavy (or light) chain immunoglobulin variable region, or afragment thereof, that is capable of binding antigen. The ability of aheavy or light chain segment to bind antigen is measured with thesegment paired with a light or heavy chain, respectively. In certainembodiments, a heavy or light chain segment that is less than a fulllength variable region will, when paired with the appropriate chain,bind with an affinity that is at least 20, 30, 40, 50, 60, 70, 80, 90,or 95% of what is observed when the full length chain is paired with alight chain or heavy chain, respectively.

An immunoglobulin variable region segment may differ from a reference orconsensus sequence. As used herein, to “differ,” means that a residue inthe reference sequence or consensus sequence is replaced with either adifferent residue or an absent or inserted residue.

The compositions and methods of the present invention encompasspolypeptides and nucleic acids having the sequences specified, orsequences substantially identical or similar thereto, e.g., sequences atleast 85%, 90%, 95% identical or higher to a specified sequence. In thecontext of an amino acid sequence, the term “substantially identical” asused herein refers to a first amino acid sequence that contains asufficient or minimum number of amino acid residues that are: i)identical to, or ii) conservative substitutions of aligned amino acidresidues in a second amino acid sequence such that the first and secondamino acid sequences can have a common structural domain and/or commonfunctional activity. For example, amino acid sequences that contain acommon structural domain having at least about 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., asequence provided herein.

In the context of nucleotide sequence, the term “substantiallyidentical” as used herein refers to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence,e.g., a sequence provided herein.

The term “functional variant” refers to polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows. Todetermine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50% or 60%, and even more preferably at least 70%,80%, 90%, or 100% of the length of the reference sequence. The aminoacid residues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. MOL. BIOL. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available on the worldwide web at gcg.com), using either a Blosum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available on the world wideweb at gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blosum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. MOL. BIOL. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecule of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) NUCLEIC ACIDS RES. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used(available on the world wide web at ncbi.nlm.nih.gov).

It is understood that the molecules of the present invention may haveadditional conservative or non-essential amino acid substitutions, whichdo not have a substantial effect on their functions.

An antibody molecule can comprise a heavy (H) chain variable region(abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, an antibody comprisestwo heavy (H) chain variable regions and two light (L) chain variableregions or an antibody binding fragment thereof. The light chains of theimmunoglobulin may be of types kappa or lambda. In one embodiment, theantibody molecule is glycosylated. An antibody molecule can befunctional for antibody-dependent cellular cytotoxicity and/orcomplement-mediated cytotoxicity, or may be non-functional for one orboth of these activities. An antibody molecule can be an intact antibodyor an antigen-binding fragment thereof.

Antibody molecules include “antigen-binding fragments” of a full lengthantibody, e.g., one or more fragments of a full-length antibody thatretain the ability to specifically bind to a target antigen of interest.Examples of antigen binding fragments encompassed within the term“antigen-binding fragment” of a full length antibody include (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′) or F(ab′)₂ fragment, a bivalent fragmentincluding two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) an scFv consisting of the VL and VH domains of a singlearm of an antibody linked together via a polypeptide linker to produce asingle chain Fv (scFv), (vi) a dAb fragment (Ward et al. (1989) NATURE341:544-546), which consists of a VH domain; and (vii) an isolatedcomplementarity determining region (CDR) that retains functionality.

As used herein, an antibody refers to a polypeptide, e.g., a tetramericor single chain polypeptide, comprising the structural and functionalcharacteristics, particularly the antigen binding characteristics, of animmunoglobulin. Typically, a human antibody comprises two identicallight chains and two identical heavy chains. Each chain comprises avariable region.

The variable heavy (VH) and variable light (VL) regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). Human antibodies have threeVH CDRs and three VL CDRs, separated by framework regions FR1-FR4. Theextent of the FRs and CDRs has been precisely defined (Kabat, E. A., etal. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTHEDITION, U. S. Department of Health and Human Services, NIH PublicationNo. 91-3242; and Chothia, C. et al. (1987) J. MOL. BIOL. 196:901-917).Each VH and VL is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxyl-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

The heavy and light immunoglobulin chains can be connected by disulfidebonds. The heavy chain constant region typically comprises threeconstant domains, CH1, CH2 and CH3. The light chain constant regiontypically comprises a CL domain. The variable region of the heavy andlight chains contains a binding domain that interacts with an antigen.The constant regions of the antibodies typically mediate the binding ofthe antibody to host tissues or factors, including various cells of theimmune system (e.g., effector cells) and the first component (Clq) ofthe classical complement system.

In yet other embodiments, the antibody molecule has a heavy chainconstant region chosen from, e.g., the heavy chain constant regions ofIgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly,chosen from, e.g., the (e.g., human) heavy chain constant regions ofIgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody moleculehas a light chain constant region chosen from, e.g., the (e.g., human)light chain constant regions of kappa or lambda. The constant region canbe altered, e.g., mutated, to modify the properties of the antibody(e.g., to increase or decrease one or more of: Fc receptor binding,antibody glycosylation, the number of cysteine residues, effector cellfunction, and/or complement function). In one embodiment the antibodyhas effector function and can fix complement. In other embodiments theantibody does not recruit effector cells or fix complement. In anotherembodiment, the antibody has reduced or no ability to bind an Fcreceptor. For example, it is an isotype or subtype, fragment or othermutant, which does not support binding to an Fc receptor, e.g., it has amutagenized or deleted Fc receptor binding region.

In one embodiment, the CD47 antibody molecule described herein comprisesan IgG4 constant region. In one embodiment, the IgG4 constant region isa wild-type constant region. In another embodiment, the IgG4 constantregion comprises a mutation, e.g., one or both of S228P and L235E, e.g.,according to EU numbering (Kabat, E. A., et al., supra). In oneembodiment, the CD47 antibody molecule described herein comprises anIgG1 constant region.

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g. altered affinity for an effectorligand, such as FcR on a cell, or the C1 component of complement can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (e.g., EP 388,151 A1,U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260). Similar types ofalterations could be described which if applied to a murine, or otherspecies immunoglobulin would reduce or eliminate these functions.

The term “immunoglobulin” comprises various broad classes ofpolypeptides that can be distinguished biochemically. Those skilled inthe art will appreciate that heavy chains are classified as gamma, mu,alpha, delta, or epsilon (γ, α, δ, ε) with some subclasses among them(e.g., γ1-γ4). It is the nature of this chain that determines the“class” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes fall within the scope of thepresent disclosure. Light chains are classified as either kappa orlambda (κ, λ). Each heavy chain class may be bound with either a kappaor lambda light chain.

As used herein, the term antibody molecule comprises intact monoclonalantibodies, polyclonal antibodies, single domain antibodies (e.g., sharksingle domain antibodies (e.g., IgNAR or fragments thereof)),multispecific antibodies (e.g., bi-specific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

Suitable antibodies include, but are not limited to, monoclonal,monospecific, polyclonal, polyspecific, human antibodies, primatizedantibodies, chimeric antibodies, bi-specific antibodies, humanizedantibodies, conjugated antibodies (e.g., antibodies conjugated or fusedto other proteins, radiolabels, or cytotoxins), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain antibodies, cameloidantibodies, and antibody fragments.

In certain embodiments, an antibody molecule is a humanized antibody. Ahumanized antibody refers to an immunoglobulin comprising a humanframework region and one or more CDRs from a non-human, e.g., mouse orrat, immunoglobulin. The immunoglobulin providing the CDRs is oftenreferred to as the “donor” and the human immunoglobulin providing theframework often called the “acceptor,” though in embodiments, no sourceor no process limitation is implied. Typically a humanized antibodycomprises a humanized light chain and a humanized heavy chainimmunoglobulin.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules Immunoglobulin domainstypically contain two beta-sheets formed of about seven beta-strands,and a conserved disulfide bond (see, e.g., A. F. Williams and A. N.Barclay (1988) ANN. REV. IMMUNOL. 6:381-405).

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence that can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that comprises an immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that interacts with the target antigen.

The antibody or antibody molecule can be derived from a mammal, e.g., arodent, e.g., a mouse or rat, horse, pig, or goat. In certainembodiments, an antibody or antibody molecule is produced using arecombinant cell. In certain embodiments, an antibody or antibodymolecule is a chimeric antibody, for example, from mouse, rat, horse,pig, or other species, bearing human constant and/or variable regionsdomains.

Multi-Specific Antibodies

In certain embodiments the antibody molecule is a multi-specificantibody molecule, e.g., it comprises a plurality of immunoglobulinvariable domains sequences, wherein a first immunoglobulin variabledomain sequence of the plurality has binding specificity for a firstepitope and a second immunoglobulin variable domain sequence of theplurality has binding specificity for a second, different epitope. In anembodiment, the first and second epitopes are present on the sameantigen, e.g., the same protein (or subunit of a multimeric protein). Inanother embodiment, the first and second epitopes overlap. In anembodiment, the first and second epitopes do not overlap. In anembodiment, the first and second epitopes are on different antigens,e.g., on different proteins (or different subunits of a multimericprotein). In another embodiment, a multi-specific antibody moleculecomprises a third, fourth or fifth immunoglobulin variable domain. In anembodiment, a multi-specific antibody molecule is a bispecific antibodymolecule, a trispecific antibody molecule, or tetraspecific antibodymolecule.

A bispecific antibody is an antibody molecule capable of bindingspecifically to two antigens. A bispecific antibody molecule ischaracterized by a first immunoglobulin variable domain sequence whichhas binding specificity for a first epitope and a second immunoglobulinvariable domain sequence that has binding specificity for a second,different epitope. The first and second epitopes can be on the sameantigen, e.g., the same protein (or subunit of a multimeric protein).The first and second epitopes may or may not overlap. In certainembodiments, the first and second epitopes are on different antigens,e.g., on different proteins (or different subunits of a multimericprotein). The bispecific antibody molecule may comprise a heavy chainvariable domain sequence and a light chain variable domain sequencewhich together define an antigen binding site with binding specificityfor a first epitope and a heavy chain variable domain sequence and alight chain variable domain sequence which together define an antigenbinding site with binding specificity for a second epitope. In oneembodiment, a bispecific antibody molecule comprises a half antibodyhaving binding specificity for a first epitope and a half antibodyhaving binding specificity for a second epitope. The bispecific antibodymolecule may comprises a half antibody, or fragment thereof, containingan antigen binding site having binding specificity for a first epitopeand a half antibody, or fragment thereof, containing an antigen bindingsite having binding specificity for a second, different epitope. In oneembodiment, a bispecific antibody molecule comprises a scFv, or fragmentthereof, have binding specificity for a first epitope and a scFv, orfragment thereof, have binding specificity for a second, differentepitope. In an embodiment, the first epitope is located on CD47 and thesecond epitope is located on CD19, CD20, CD38, or the HER2/neu receptor.

Anti-CD47 Antibody Molecules

The present invention provides isolated, recombinant and/or syntheticanti-CD47 human, primate, rodent, mammalian, chimeric, humanized and/orCDR-grafted antibodies as well as compositions and encoding nucleic acidmolecules comprising at least one polynucleotide encoding at least aportion of one anti-CD47 antibody molecule. The present inventionfurther includes, but is not limited to, methods of making and usingsuch nucleic acids and antibodies including diagnostic and therapeuticcompositions, methods and devices.

The terms “isolated protein” or “isolated antibody molecule” referred toherein means a protein or antibody molecule, which by virtue of itsorigin or source of derivation (1) is not associated with proteins foundin nature, (2) is free of other proteins from the same source, (3) isexpressed by a cell from a different species, or (4) does not occur innature.

Exemplary antibody molecules of the invention include the 2.3D11antibody having a variable heavy chain region (VH) and/or variable light(VL) chain region, heavy chain CDR1, CD2, and CD3, light chain CDR1,CD2, and CDR3, and full heavy and light chains, as shown in thesequences below.

Antibody 2.3D11

As shown in the Examples, it has been discovered that antibody 2.3D11 isa novel antibody that is capable of interrupting the interaction betweenCD47 and SIRPα without inducing significant hemagglutination oferythrocytes. The sequences of the individual heavy and light chainvariable regions of the 2.3D11 antibody, and antibody moleculescontaining such variable region sequences are described below.

Variable Heavy Chain (VH) with leader sequence:

(SEQ ID NO: 3) MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSGTLSLTCAVSGVSIRSINWWNWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLNSVTAADTAVYYCARDGGIAVTDYYYYGLDVWGQGTTVTVSS.

Variable Heavy Chain (VH) without leader sequence:

(SEQ ID NO: 4) QVQLQESGPGLVKPSGTLSLTCAVSGVSIRSINWWNWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLNSVTAADTAVYYCARDGGIAVTDYYYYGLDVWGQGTTVTVSS.

Variable Light Chain (VL) with leader sequence:

(SEQ ID NO: 5) MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASESVSSNLAWYQQKPGQAPRLLIYGAFNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWFTFGGGTKVEIK.

Variable Light Chain (VL) without leader sequence:

(SEQ ID NO: 6) EIVLTQSPATLSLSPGERATLSCRASESVSSNLAWYQQKPGQAPRLLIYGAFNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSDWFTFGGG TKVEIK.

VH Complementarity Determining Region 1 (VH CDR1):

SINWWN (SEQ ID NO: 7).

VH Complementarity Determining Region 2 (VH CDR2):

EIYHSGSTNYNPSLKS (SEQ ID NO: 8).

VH Complementarity Determining Region 3 (VH CDR3):

DGGIAVTDYYYYGLDV (SEQ ID NO: 9).

VL Complementarity Determining Region 1 (VL CDR1):

RASESVSSNLA (SEQ ID NO: 10).

VL Complementarity Determining Region 2 (VL CDR2):

GAFNRAT (SEQ ID NO: 11).

VL Complementarity Determining Region 3 (VL CDR3):

QQRSDWFT (SEQ ID NO: 12).

Full Heavy Chain Sequence without leader sequence, including anannotation of the framework regions 1-4 (FR1-FR4), complementarydetermining regions 1-3 (CDR1-CDR3) and constant region(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region):

Full Heavy Chain Sequence with leader sequence (Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region):

Full Light Chain Sequence without leader sequence including anannotation of the framework regions 1-4 (FR1-FR4), complementarydetermining regions 1-3 (CDR1-CDR3) and constant region(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region):

Full Light Chain Sequence with leader sequence: (Leadersequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region):

In certain embodiments, an exemplary antibody of the invention comprisesa heavy chain variable domain with complementary determining sequencesCDR1-3 corresponding to residues 31-36, 51-66, and 99-114, respectively,of SEQ ID NO: 4. In certain embodiments, an exemplary antibody of theinvention comprises a heavy chain variable domain with frameworksequences FR1-FR4 corresponding to residues 1-30, 37-50, 67-98, and115-125, respectively, of SEQ ID NO: 4. In certain embodiments, anexemplary antibody of the invention comprises a light chain variabledomain with complementary determining sequences CDR1-3 corresponding toresidues 24-34, 50-56, and 89-96, respectively, of SEQ ID NO: 6. Incertain embodiments, an exemplary antibody of the invention comprises alight chain variable domain with framework sequences FR1-FR4corresponding to residues 1-23, 35-49, 57-88, and 97-106, respectively,of SEQ ID NO: 6.

In certain embodiments, it is contemplated that a heavy chain variableregion sequence, for example, the VH sequence of SEQ ID NO: 4, may becovalently linked to a variety of heavy chain constant region sequencesknown in the art. Similarly, it is contemplated that a light chainvariable region sequence, for example, the VL of SEQ ID NO: 6, maybe becovalently linked to a variety of light chain constant region sequencesknown in the art. For example, the heavy chain variable region sequencemay be used with a heavy chain constant region sequence derived from anIgG1, IgG2, IgG3, or IgG4 molecule.

In certain embodiments, the constant region of the heavy chain of theantibody is of human IgG1 isotype, having an amino acid sequence:

In certain embodiments, the human IgG1 constant region is modified atamino acid Asn297 (Boxed) to prevent to glycosylation of the antibody,for example Asn297Ala (N297A). In certain embodiments, the constantregion of the antibody is modified at amino acid Leu235 (Boxed) to alterFc receptor interactions, for example Leu235Glu (L235E) or Leu235Ala(L235A). In certain embodiments, the constant region of the antibody ismodified at amino acid Leu234 (Boxed) to alter Fc receptor interactions,e.g., Leu234Ala (L234A). In certain embodiments, the constant region ofthe antibody is modified at amino acid Glu233 (Boxed), e.g., Glu233Pro(E233P). In some embodiments, the constant region of the antibody isaltered at both amino acid 234 and 235, for example Leu234Ala andLeu235Ala (L234A/L235A). In certain embodiments, the constant region ofthe antibody is altered at amino acids 233, 234, and 234, for example,Glu233Pro, Leu234Ala, and Leu235Ala (E233P L234A/L235A) (Armour K L. etal. (1999) EUR. J. IMMUNOL. 29(8):2613-24). All residue numbers areaccording to EU numbering (Kabat, E. A., et al., supra).

In certain embodiments, the constant region of the heavy chain of theantibody is of human IgG2 isotype, having an amino acid sequence:

In certain embodiments, the human IgG2 constant region is modified atamino acid Asn297 (Boxed) to prevent to glycosylation of the antibody,e.g., Asn297Ala (N297A), where the residue numbers are according to EUnumbering (Kabat, E. A., et al., supra).

hl certain embodiments, the constant region of the heavy chain of theantibody is of human IgG3 isotype, having an amino acid sequence:

In certain embodiments, the human IgG3 constant region is modified atamino acid Asn297 (Boxed) to prevent to glycosylation of the antibody,e.g., Asn297Ala (N297A). In some embodiments, the human IgG3 constantregion is modified at amino acid Arg435 (Boxed) to extend the half-life,e.g., Arg435H (R435H). All residue numbers are according to EU numbering(Kabat, E. A., et al., supra).

In certain embodiments, the constant region of the heavy chain of theantibody is of human IgG4 isotype, having an amino acid sequence:

In certain embodiments, human IgG4 constant region is modified withinthe hinge region to prevent or reduce strand exchange, e.g., in someembodiments human IgG4 constant region is modified at Ser228 (Boxed),e.g., Ser228Pro (S228P). In other embodiments, the human IgG4 constantregion is modified at amino acid Leu235 (Boxed) to alter Fc receptorinteractions, e.g., Leu235Glu (L235E). In some embodiments, the humanIgG4 constant region is modified at both Ser228 and Leu335, e.g.,Ser228Pro and Leu235Glu (S228P/L235E), and comprises the amino acidsequence of SEQ ID NO: 21. In some embodiments, the human IgG4 constantregion is modified at amino acid Asn297 (Boxed) to prevent toglycosylation of the antibody, e.g., Asn297Ala (N297A). All residuenumbers are according to EU numbering (Kabat, E. A., et al., supra).

In certain embodiments, the constant region of the heavy chain of theantibody is of human IgM isotype, having an amino acid sequence:

(SEQ ID NO: 33) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY.

In certain embodiments, the human IgG constant region is modified toenhance FcRn binding. Examples of Fc mutations that enhance binding toFcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E,respectively) (Dall'Acqua et al. (2006) J. BIOL. CHEM. 281(33):23514-23524), or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al.(2010) NATURE BIOTECH. 28(2): 157-159). All residue numbers areaccording to EU numbering (Kabat, E. A., et al., supra).

In some embodiments, the human IgG constant region is modified to alterantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), e.g., the amino acidmodifications described in Natsume et al. (2008) CANCER RES. 68(10):3863-72; Idusogie et al. (2001) J. IMMUNOL. 166(4): 2571-5; Moore et al.(2010) MABS 2(2): 181-189; Lazar et al. (2006) PROC. NATL. ACAD. SCI.USA 103(11): 4005-4010, Shields et al. (2001) J. BIOL. CHEM. 276(9):6591-6604; Stavenhagen et al. (2007) CANCER RES. 67(18): 8882-8890;Stavenhagen et al. (2008) ADVAN. ENZYME REGUL. 48: 152-164; Alegre etal. (1992) J. IMMUNOL. 148: 3461-3468.

In some embodiments, the human IgG constant region is modified to induceheterodimerization. For example, a heavy chain having an amino acidmodification within the CH3 domain at Thr366, e.g., a substitution witha more bulky amino acid, e.g., Try (T366W), is able to preferentiallypair with a second heavy chain having a CH3 domain having amino acidmodifications to less bulky amino acids at positions Thr366, Leu368, andTyr407, e.g., Ser, Ala and Val, respectively (T366S/L368A/Y407V).Heterodimerization via CH3 modifications can be further stabilized bythe introduction of a disulfide bond, for example by changing Ser354 toCys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (see, Carter(2001) J. IMMUNOL. METHODS 248: 7-15).

In certain embodiments, the constant region of the light chain of theantibody is a human kappa constant region having an amino acid sequence:

(SEQ ID NO: 22) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC.

In certain embodiments, the constant region of the light chain of theantibody is a human lambda constant region having an amino acidsequence:

(SEQ ID NO: 34) GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP TEC.

In certain embodiments, an exemplary antibody of the invention comprisesa heavy chain variable domain of the 2.3D11 antibody and a human IgG1heavy chain constant domain depicted as follows(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region; without the leadersequence):

In certain embodiments, an exemplary antibody of the invention comprisesa heavy chain variable domain of the 2.3D11 antibody and a human IgG4heavy chain constant domain depicted as follows(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region; without the leadersequence):

In certain embodiments, an exemplary antibody of the invention comprisesa heavy chain variable domain of the 2.3D11 antibody and a human IgG4heavy chain constant domain with Ser228Pro and Leu235Glu substitutionsdepicted as follows (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region;without the leader sequence, mutated residues boxed):

In certain embodiments, an exemplary antibody of the invention comprisesa light chain variable domain of the 2.3D11 antibody and a human kappaconstant domain depicted as follows(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region; without the leadersequence):

In certain embodiments, the anti-CD47 antibody molecule comprises one orboth of (a) and (b), wherein (a) and (b) are as follows:

(a)(i) light chain CDR1, CDR2 and CDR3, e.g., Chothia or Kabat lightchain CDRs, from SEQ ID NO: 16,

(a)(ii) light chain CDR1 of SEQ ID NO: 10, light chain CDR2 of SEQ IDNO: 11, and light chain CDR3 of SEQ ID NO: 12,

(a)(iii) light chain CDRs CDR1, CDR2 and CDR3, that collectively, differby no more than 1, 2, 3, 4, 5, or 6 amino acid residues from the lightchain CDRs of (a)(i) and (a)(ii);

(a)(iv) a light chain variable region of SEQ ID NO: 6;

(a)(v) an antigen binding fragment of SEQ ID NO: 6;

(a)(vi) an amino acid sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, residues from the sequence of (a)(iv) or a)(v);

(a)(vii) an amino acid sequence that is substantially identical (e.g.,at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical) to the sequenceof (a)(iv) or (a)(v); and

(b)(i) heavy chain CDR1, CDR2 and CDR3, e.g., Chothia or Kabat heavychain CDRs, from SEQ ID NO: 15,

(b)(ii) heavy chain CDR1 of SEQ ID NO: 7, heavy chain CDR2 of SEQ ID NO:8, and heavy chain CDR3 of SEQ ID NO: 9,

(b)(iii) heavy chain CDRs CDR1, CDR2 and CDR3, that collectively, differby no more than 1, 2, 3, 4, 5, or 6 amino acid residues from the heavychain CDRs of (b)(i) and (b)(ii);

(b)(iv) a heavy chain variable region of SEQ ID NO: 4;

(b)(v) an antigen binding fragment of SEQ ID NO: 4;

(b)(vi) an amino acid sequence that differs by no more than 1, 2, 3, 4,5, 6, 7, 8, 9, or 10, residues from the sequence of (b)(iv) or (b)(v);and

(b)(vii) an amino acid sequence that is substantially identical (e.g.,at least 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical) to the sequenceof (b)(iv) or (b)(v).

In certain configurations, the antibody molecule comprises: (a)(i) andany one of (b); (a)(ii) and any one of (b); (a)(iii) and any one of (b);(a)(iv) and any one of (b); (a)(v) and any one of (b); (a)(vi) and anyone of (b); (a)(vii) and any one of (b); (b)(i) and any one of (a);(b)(ii) and any one of (a); (b)(iii) and any one of (a); (b)(iv) and anyone of (a); (b)(v) and any one of (a); (b)(vi) and any one of (a);(b)(vii) and any one of (a). In certain configurations, the antibodymolecule comprises: (a)(i) and (b)(i); (a)(ii) and (b)(ii); (a)(iii) and(b)(iii); (a)(iv) and (b)(iv); (a)(v) and (b)(v); or (a)(vi) and(b)(vi).

It is contemplated that, with respect to certain of the uses, forexample, therapeutic interventions described herein, the anti-CD47antibody having little or no hemagglutination activity includes one ormore of the antibodies described herein, for example, the 2.3D11antibody and variants thereof, as well as the antibodies known in theart to bind CD47 and disrupt the CD47-SIRPα interaction with little orno hemagglutination activity, including the antibodies described in U.S.Pat. No. 9,045,541, including, for example, the antibodies referred toas 2A1, 2A1-xi, AB6.12, AB6.12-IgG1, AB6.12-IgG4P and AB6.12-IgG4PE. Forexample antibody AB6.12 comprises the variable heavy chain sequence ofSEQ ID NO: 11 and the variable light chain sequence of SEQ ID NO: 42 asset forth in Table 1 of U.S. Pat. No. 9,045,541 (corresponding to SEQ IDNOs. 27 and 28, respectively, as disclosed herein). An additionalexemplary antibody is the anti-CD47 antibody, 5F9G4, which comprises avariable heavy chain of SEQ ID NO: 29 and a variable light chain of SEQID NO: 30, and is described in Liu et al. (2016) PLoS ONE10(9):e0137345.

The antibody molecules described herein may have minor variations in theamino acid sequences compared to a reference, and, for example, may haveat least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity relative to areference sequence, for example, the heavy chain of SEQ ID NO: 15 or thelight chain of SEQ ID NO: 16. The mutations may include conservativeamino acid substitutions, which are substitutions that take place withina family of amino acids related in their side chains, for example,aspartic acid and glutamic acid.

Antibody molecules of the present invention can be expressed in amodified form. For instance, a region of additional amino acids,particularly charged amino acids, can be added to the N-terminus of anantibody molecule to improve stability and persistence in the host cell,during purification, or during subsequent handling and storage. Also,peptide moieties can be added to an antibody molecule of the presentinvention to facilitate purification. Such regions can be removed priorto final preparation of an antibody molecule or at least one fragmentthereof. Such methods are described in many standard laboratory manuals,such as Sambrook, supra; Ausubel, et al., ed., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, Inc., NY, N.Y. (1987-2001).

It is contemplated that the antibodies provided may be used in thegeneration of anti-idiotype antibodies thereto, as well as compositionscomprising an anti-idiotype antibody molecule and encoding nucleic acidmolecules comprising at least one polynucleotide encoding at least aportion of an anti-idiotype antibody molecule.

The antibody molecules bind CD47 with an equilibrium binding constant of≦1 μM, e.g., ≦100 nM, preferably ≦10 nM, and more preferably ≦1 nM, asmeasured using standard binding assays, for example, the BIACore-basedbinding assay.

Antibody molecules of the present invention may be characterizedrelative to a reference anti-CD47 antibody, for example, B6H12, 2D3,MABL, CC2C6, or BRIC126. Antibody B6H12 is described, for example, inU.S. Pat. Nos. 5,057,604 and 9,017,675, is commercially available fromAbcam, PLC, Santa Cruz Biotechnology, Inc., and eBioscience, Inc., andcomprises a heavy chain variable region of SEQ ID NO: 31 and a lightchain variable region of SEQ ID NO: 32. Antibody MABL is described, forexample, in Uno S, Kinoshita Y, Azuma Y et al. (2007) ONCOL. REP. 17:1189-94, and Kikuchi Y, Uno S, Yoshimura Y et al. (2004) BIOCHEM.BIOPHYS. RES. COMMUN. 315: 912-8. Antibody CC2C6 is described, forexample, in Martina Seiffert et al. (1997) BLOOD 94(11): 3633-3643, andis commercially available from Santa Cruz Biotechnology, Inc. AntibodyBRIC126 is described, for example, in Avent et al. (1988) BIOCHEM. J.251: 499-505. Antibody 2D3 is commercially available from eBioscience,Inc., and unlike the other reference antibodies does not interfere withthe binding between CD47 and SIRPα.

Antibody Molecule Expression

Nucleic acids of the present invention can be expressed in a host cellthat contains endogenous DNA encoding an antibody molecule of thepresent invention. Such methods are well known in the art, e.g., asdescribed in U.S. Pat. Nos. 5,580,734, 5,641,670, 5,733,746, and5,733,761. Also see, e.g., Sambrook, et al., supra, and Ausubel, et al.,supra. Those of ordinary skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. Illustrative of cellcultures useful for the production of the antibody molecules, specifiedportions or variants thereof, are mammalian cells. Mammalian cellsystems often will be in the form of monolayers of cells althoughmammalian cell suspensions or bioreactors can also be used. A number ofsuitable host cell lines capable of expressing intact glycosylatedproteins have been developed in the art, and include the COS-1 (e.g.,ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCCCRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) celllines, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, HeLa cells and the like,which are readily available from, for example, American Type CultureCollection, Manassas, Va. Yeast and bacterial host cells may also beused and are well known to those of skill in the art. Other cells usefulfor production of nucleic acids or proteins of the present invention areknown and/or available, for instance, from the American Type CultureCollection Catalogue of Cell Lines and hybridomas or other known orcommercial sources.

Expression vectors can include one or more of the following expressioncontrol sequences, such as, but not limited to an origin of replication;a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S.Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk(phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Pat.No. 5,266,491), at least one human immunoglobulin promoter; an enhancer,and/or processing information sites, such as ribosome binding sites, RNAsplice sites, polyadenylation sites (e.g., an SV40 large T Ag poly Aaddition site), and transcriptional terminator sequences). See, e.g.,Ausubel et al., supra; Sambrook, et al., supra.

Expression vectors optionally include at least one selectable marker.Such markers include, e.g., but are not limited to, methotrexate (MTX),dihydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216; 4,634,665;4,656,134; 4,956,288; 5,149,636; 5,179,017), ampicillin, neomycin(G418), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos.5,122,464; 5,770,359; and 5,827,739), resistance for eukaryotic cellculture, and tetracycline or ampicillin resistance genes for culturingin E. coli and other bacteria or prokaryotes. Appropriate culture mediaand conditions for the above-described host cells are known in the art.Suitable vectors will be readily apparent to the skilled artisan.Introduction of a vector construct into a host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other known methods. Such methods are described in the art,such as Sambrook, supra; Ausubel, supra.

The nucleic acid insert should be operatively linked to an appropriatepromoter. The expression constructs will further contain sites fortranscription initiation, termination and, in the transcribed region, aribosome binding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al. (1983) J.VIROL. 45:773-781). Additionally, gene sequences to control replicationin the host cell can be incorporated into the vector, as known in theart.

Antibody Molecule Isolation and Purification

Antibody molecules described herein can be recovered and purified fromrecombinant cell cultures by well-known methods including, but notlimited to, protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (HPLC) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,New York, N.Y., (1997-2001).

Antibody molecules described herein can include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a eukaryotic host, including,for example, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, theantibody molecule of the present invention can be glycosylated or can benon-glycosylated, with glycosylated preferred. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra;Ausubel, supra, Colligan, Protein Science, supra.

Nucleic Acid Molecules

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, as CDR1, CDR2 and/or CDR3 ofat least one heavy chain (e.g., SEQ ID NOs: 7-9) or light chain (e.g.,SEQ ID NOs: 10-12); nucleic acid molecules comprising the codingsequence for an anti-CD47 antibody molecule or variable region (e.g.,SEQ ID NOs: 4 and 6); and nucleic acid molecules which comprise anucleotide sequence substantially different from those described abovebut which, due to the degeneracy of the genetic code, still encode atleast one anti-CD47 antibody molecule as described herein and/or asknown in the art. Given that the genetic code is well known in the art,it is routine for one skilled in the art to generate such degeneratenucleic acid variants that code for specific anti-CD47 antibodymolecules of the present invention. See, e.g., Ausubel, et al., supra,and such nucleic acid variants are included in the present invention. Incertain embodiments, a nucleic acid molecule encoding a heavy chainvariable domain of the 2.3D11 antibody and a human IgG1 heavy chainconstant domain comprises SEQ ID NO: 35. In certain embodiments, anucleic acid molecule encoding a heavy chain variable domain of the2.3D11 antibody and a human IgG4 heavy chain constant domain comprisesSEQ ID NO: 36. In certain embodiments, a nucleic acid molecule encodinga heavy chain variable domain of the 2.3D11 antibody and a human IgG4heavy chain constant domain with Ser228Pro and Leu235Glu substitutionscomprises SEQ ID NO: 37. In certain embodiments, a nucleic acid moleculeencoding a light chain variable domain of the 2.3D11 antibody and ahuman kappa constant domain comprises SEQ ID NO: 38

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-CD47 antibody moleculecan include, but are not limited to, those encoding the amino acidsequence of an antibody fragment, by itself; the coding sequence for theentire antibody or a portion thereof; the coding sequence for anantibody, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example—ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding an antibody molecule can be fused to a marker sequence, such asa sequence encoding a peptide that facilitates purification of the fusedantibody molecule comprising an antibody molecule fragment or portion.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art. Thenucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention. Additional sequences can beadded to such cloning and/or expression sequences to optimize theirfunction in cloning and/or expression, to aid in isolation of thepolynucleotide, or to improve the introduction of the polynucleotideinto a cell. Use of cloning vectors, expression vectors, adapters, andlinkers is well known in the art. (See, e.g., Ausubel, supra; orSambrook, supra)

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Antibody Molecule Compositions

For therapeutic use, an antibody preferably is combined with apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means buffers, carriers, and excipients suitable foruse in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio. Thecarrier(s) should be “acceptable” in the sense of being compatible withthe other ingredients of the formulations and not deleterious to therecipient. Pharmaceutically acceptable carriers include buffers,solvents, dispersion media, coatings, isotonic and absorption delayingagents, and the like, that are compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is known in the art.

Accordingly, antibody molecule compositions of the present invention cancomprise at least one of any suitable excipients, such as, but notlimited to, diluent, binder, stabilizer, buffers, salts, lipophilicsolvents, preservative, adjuvant or the like. Pharmaceuticallyacceptable excipients are preferred. Non-limiting examples of, andmethods of preparing such sterile solutions are well known in the art,such as, but not limited to, those described in Gennaro, Ed.,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Mack Publishing Co.(Easton, Pa.) 1990. Pharmaceutically acceptable carriers can beroutinely selected that are suitable for the mode of administration,solubility and/or stability of the antibody molecule, fragment orvariant composition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody molecule components,which can also function in a buffering capacity, include alanine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Antibody molecule compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, acetic acid, ascorbic acid, gluconic acid,carbonic acid, tartaric acid, succinic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers.

Additionally, antibody molecule compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the antibody molecule compositions according to theinvention are known in the art, e.g., as listed in “REMINGTON: THESCIENCE & PRACTICE OF PHARMACY”, 19th ed., Williams & Williams, (1995),and in the “PHYSICIAN'S DESK REFERENCE”, 52nd ed., Medical Economics,Montvale, N.J. (1998). Preferred carrier or excipient materials arecarbohydrates (e.g., saccharides and alditols) and buffers (e.g.,citrate) or polymeric agents.

The present invention provides for stable compositions, comprising atleast one anti-CD47 antibody molecule in a pharmaceutically acceptableformulation. Preserved formulations contain at least one knownpreservative or optionally selected from the group consisting of atleast one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzylalcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde,chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,benzethonium chloride, sodium dehydroacetate and thimerosal, or mixturesthereof in an aqueous diluent. Any suitable concentration or mixture canbe used as known in the art, such as 0.001-5%, or any range or valuetherein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01,0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or valuetherein. Non-limiting examples include, no preservative, 0.1-2% m-cresol(e.g., 0.2, 0.3, 0.4, 0.5, 0.9, or 1.0%), 0.1-3% benzyl alcohol (e.g.,0.5, 0.9, 1.1, 1.5, 1.9, 2.0, or 2.5%), 0.001-0.5% thimerosal (e.g.,0.005 or 0.01%), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, or1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, or 1.0%), and the like.

Pharmaceutical compositions containing antibodies disclosed herein canbe presented in a dosage unit form and can be prepared by any suitablemethod. A pharmaceutical composition should be formulated to becompatible with its intended route of administration. Examples of routesof administration are intravenous (IV), intradermal, inhalation,transdermal, topical, transmucosal, and rectal administration. Apreferred route of administration for monoclonal antibodies is IVinfusion. Useful formulations can be prepared by methods known in thepharmaceutical art. For example, see REMINGTON'S PHARMACEUTICAL SCIENCES(1990) supra. Formulation components suitable for parenteraladministration include a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as EDTA; buffers such as acetates,citrates or phosphates; and agents for the adjustment of tonicity suchas sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol), and suitable mixturesthereof.

In certain embodiments, the pharmaceutically acceptable compositioncomprises the anti-CD47 antibody in 10 mM histidine, 280 mM sucrose, and0.01% TWEEN 80 at pH 6.0

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished by any suitable method, e.g., filtration through sterilefiltration membranes. Where the composition is lyophilized, filtersterilization can be conducted prior to or following lyophilization andreconstitution.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, and liposomes. The preferred form depends on theintended mode of administration and therapeutic application. Typicalpreferred compositions are in the form of injectable or infusiblesolutions. The preferred mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular).In a preferred embodiment, the preparation is administered byintravenous infusion or injection. In another preferred embodiment, thepreparation is administered by intramuscular or subcutaneous injection.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, subcutaneous, intraarterial,intrathecal, intracapsular, intraorbital, intravitreous, intracardiac,intradermal, intraperitoneal, transtracheal, inhaled, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

Articles of Manufacture

The present invention provides an article of manufacture, comprisingpackaging material and at least one vial comprising a solution of atleast one anti-CD47 antibody molecule with the prescribed buffers and/orpreservatives, optionally in an aqueous diluent. The aqueous diluentoptionally further comprises a pharmaceutically acceptable preservative.Preservatives include those selected from the group consisting ofphenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof. The concentration of preservative used in theformulation is a concentration sufficient to yield an anti-microbialeffect. Such concentrations are dependent on the preservative selectedand are readily determined by the skilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4.0 to about pH 10.0, from about pH5.0 to about pH 9.0, or about pH 6.0 to about pH 8.0.

Other additives, such as a pharmaceutically acceptable solubilizers likeTWEEN 20 (polyoxyethylene (20) sorbitan monolaurate), TWEEN 40(polyoxyethylene (20) sorbitan monopalmitate), TWEEN 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

Therapeutic Applications

In addition, the invention provides methods of treating disordersassociated with elevated levels of CD47 expression in certain celltypes, for example, certain cancers, whose cells exhibit elevated levelsof CD47 expression. As a result, the invention provides a method oftreating a subject, for example, a subject with a cancer, in needthereof. The method comprises administering an effective amount of ananti-CD47 antibody or a composition comprising an anti-CD47 antibody tothe subject in need thereof.

As used herein, the terms “subject” and “patient” refer to organisms tobe treated by the methods of the present invention. Such organismspreferably include, but are not limited to, mammals (e.g., murines,simians, equines, bovines, porcines, canines, felines, and the like),and more preferably includes humans. As used herein, the terms, “treat,”“treatment” and “treating” includes any effect, e.g., lessening,reducing, modulating, ameliorating or eliminating, that results in theimprovement of the condition, disease, disorder, and the like, orameliorating a symptom thereof.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., an anti-CD47 antibody molecule) sufficient to effectbeneficial or desired results. An effective amount can be administeredin one or more administrations, applications or dosages and is notintended to be limited to a particular formulation or administrationroute. Generally, a therapeutically effective amount of active componentis in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1mg/kg to 10 mg/kg. The dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; the age, health, andweight of the recipient; the type and extent of disease or indication tobe treated, the nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. The initialdosage can be increased beyond the upper level in order to rapidlyachieve the desired blood-level or tissue-level. Alternatively, theinitial dosage can be smaller than the optimum, and the daily dosage maybe progressively increased during the course of treatment. Human dosagecan be optimized, e.g., in a conventional Phase I dose escalation studydesigned to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary,depending on factors such as route of administration, dosage amount,serum half-life of the antibody, and the disease being treated.Exemplary dosing frequencies are once per day, once per week and onceevery two weeks. Formulation of monoclonal antibody-based drugs iswithin ordinary skill in the art. In some embodiments, a monoclonalantibody is lyophilized, and then reconstituted in buffered saline, atthe time of administration

The present invention provides a method for treating cancer in a cell,tissue, organ, animal or patient. Examples of cancers include, but arenot limited to, solid tumors, soft tissue tumors, hematopoietic tumorsand metastatic lesions. Examples of hematopoietic tumors include,leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell,T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocyticleukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformedCLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairycell leukemia, myelodysplastic syndrome (MDS), a lymphoma, Hodgkin'sdisease, a malignant lymphoma, non-Hodgkin's lymphoma, Burkitt'slymphoma, multiple myeloma, or Richter's Syndrome (Richter'sTransformation). Examples of solid tumors include malignancies, e.g.,sarcomas, adenocarcinomas, and carcinomas, of the various organ systems,such as those affecting head and neck (including pharynx), thyroid, lung(small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid,gastrointestinal (e.g., oral, esophageal, stomach, liver, pancreas,small intestine, colon and rectum, anal canal), genitals andgenitourinary tract (e.g., renal, urothelial, bladder, ovarian, uterine,cervical, endometrial, prostate, testicular), CNS (e.g., neural or glialcells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma). Incertain embodiments, the solid tumor is NMDA receptor positive teratoma.In certain embodiments, the cancer is chosen from breast cancer, coloncancer, pancreatic cancer (e.g., pancreatic neuroendocrine tumors(PNETs) or pancreatic ductal adenocarcinoma (PDAC)), stomach, uterinecancer, or ovarian cancer.

In one embodiment, the cancer is a cancer associated with ascites.Ascites is a symptom of many types of cancer and can also be caused by anumber of conditions, such as advanced liver disease. The types ofcancer that are likely to cause ascites are cancer of the breast, lung,large bowel (colon), stomach, pancreas, ovary, womb (endometrium) andthe peritoneum. In some embodiments, the cancer associated with ascitesis chosen from breast cancer, colon cancer, pancreatic cancer, stomach,uterine cancer, or ovarian cancer. In some embodiments, the cancer isassociated with pleural effusions, e.g., lung cancer.

Additional hematological cancers include, Myelodysplastic syndrome (MDS)(e.g., preleukemia, refractory anemias, Ph-negative chronic myelocyticleukemia, chronic myelomonocytic leukemia, myeloid metaplasia),Non-Hodgkin lymphoma (e.g., diffuse large B cell lymphoma, chroniclymphocytic leukemia, mantle cell lymphoma, B lymphoblasticleukemia/lymphoma, peripheral T cell lymphoma and Burkitt's lymphoma),B-lymphoblastic leukemia/lymphoma; B-cell chronic lymphocyticleukemia/small lymphocytic lymphoma; B-cell prolymphocytic leukemia;Lymphoplasmacytic lymphoma; Splenic marginal zone B-cell lymphoma(±villous lymphocytes); Hairy cell leukemia; Plasma cellmyeloma/plasmacytoma; Extranodal marginal zone B-cell lymphoma of theMALT type; Nodal marginal zone B-cell lymphoma (±monocytoid B cells);Follicular lymphoma; Mantle cell lymphoma; Diffuse large B-celllymphomas; Burkitt's lymphoma; Precursor T-lymphoblasticlymphoma/leukemia; T-cell prolymphocytic leukemia; T-cell granularlymphocytic leukemia; Aggressive NK cell leukemia; Adult T-celllymphoma/leukemia (HTLV 1-positive); Extranodal NK/T-cell lymphoma,nasal type; Enteropathy-type T-cell lymphoma; Hepatosplenic γ-δ T-celllymphoma; Subcutaneous panniculitis-like T-cell lymphoma; Mycosisfungoides/Sezary syndrome; Anaplastic large cell lymphoma, T/null cell,primary cutaneous type; Anaplastic large cell lymphoma, T-/null-cell,primary systemic type; Peripheral T-cell lymphoma, not otherwisecharacterized; Angioimmunoblastic T-cell lymphoma, chronic lymphocyticleukemia (CLL), chronic myelocytic leukemia (CML), multiple myeloma,polycythemia vera or myelofibrosis, cutaneous T-cell lymphoma, smalllymphocytic lymphoma (SLL), marginal zone lymphoma, CNS lymphoma,immunoblastic large cell lymphoma, and precursor B-lymphoblasticlymphoma.

Anti CD-47 antibodies, including, e.g., the antibody molecules describedherein, can also be used to treat disorders associated with cancer,e.g., cancer-induced encephalopathy

Anti CD-47 antibodies, including, e.g., the antibody molecules describedherein, can also be used to treat inflammatory, autoimmune, fibrotic,fibroproliferative, atopic, or angiogenic disorders. Examples ofinflammatory disorders include but are not limited to chronicobstructive pulmonary disease, asthma, rheumatoid arthritis,inflammatory bowel disease (including Crohn's disease and ulcerativecolitis), multiple sclerosis, psoriasis, ischemia-reperfusion injuries,septic shock, age-related macular degeneration (e.g., wet age-relatedmacular degeneration), atherosclerosis, Alzheimer's disease, Parkinson'sdisease, cardiovascular disease, vasculitis, type I and II diabetes,metabolic syndrome, diabetic retinopathy, restenosis. Examples ofautoimmune diseases include but are not limited to asthma, rheumatoidarthritis, inflammatory bowel disease, multiple sclerosis, psoriasis,type I diabetes, systemic lupus erythematosus (SLE), Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, Guillain-Barré syndrome,autoimmune hepatitis, and Myasthenia gravis. Examples of fibroticdiseases include but are not limited to scleroderma, liver fibrosis,pancreatic fibrosis, chronic obstructive pulmonary disease, diabeticnephropathy, sarcoidosis, idiopathic pulmonary fibrosis, cirrhosis,cystic fibrosis, neurofibromatosis, endometriosis, post-operativefibroids, and restenosis. Examples of atopic disease include but are notlimited to atopic dermatitis, atopic asthma, and allergic rhinitis.

The methods and compositions of the invention can be used in combinationwith other therapeutic agents and/or modalities. The term administered“in combination,” as used herein, is understood to mean that two (ormore) different treatments are delivered to the subject during thecourse of the subject's affliction with the disorder, such that theeffects of the treatments on the patient overlap at a point in time. Incertain embodiments, the delivery of one treatment is still occurringwhen the delivery of the second begins, so that there is overlap interms of administration. This is sometimes referred to herein as“simultaneous” or “concurrent delivery.” In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

In one embodiment, the methods of the invention include administering tothe subject an anti-CD47 molecule, e.g., an anti-CD47 antibody moleculedescribed herein, e.g., a composition or preparation, described herein,in combination with one or more additional therapies, e.g., surgery,radiation therapy, or administration of another therapeutic preparation.In one embodiment, the additional therapy may include chemotherapy,e.g., a cytotoxic agent. In one embodiment the additional therapy mayinclude a targeted therapy, e.g. a tyrosine kinase inhibitor, aproteasome inhibitor, or a protease inhibitor. In one embodiment, theadditional therapy may include an anti-inflammatory, anti-angiogenic,anti-fibrotic, or anti-proliferative compound, e.g., a steroid, abiologic immunomodulator, a monoclonal antibody, an antibody fragment,an aptamer, an siRNA, an antisense molecule, a fusion protein, acytokine, a cytokine receptor, a bronchodialator, a statin, ananti-inflammatory agent (e.g. methotrexate), or an NSAID. In anotherembodiment, the additional therapy could include combining therapeuticsof different classes. The polysaccharide preparation and the additionaltherapy can be administered simultaneously or sequentially.

Exemplary cytotoxic agents that can be administered in combination withthe polysaccharide preparation include antimicrotubule agents,topoisomerase inhibitors, antimetabolites, protein synthesis anddegradation inhibitors, mitotic inhibitors, alkylating agents,platinating agents, inhibitors of nucleic acid synthesis, histonedeacetylase inhibitors (HDAC inhibitors, e.g., vorinostat (SAHA,MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A(TSA), mocetinostat (MGCD0103), belinostat (PXD101), romidepsin (FK228,depsipeptide)), DNA methyltransferase inhibitors, nitrogen mustards,nitrosoureas, ethylenimines, alkyl sulfonates, triazenes, folateanalogs, nucleoside analogs, ribonucleotide reductase inhibitors, vincaalkaloids, taxanes, epothilones, intercalating agents, agents capable ofinterfering with a signal transduction pathway, agents that promoteapoptosis and radiation, or antibody molecule conjugates that bindsurface proteins to deliver a toxic agent. In one embodiment, thecytotoxic agent that can be administered with a preparation describedherein is a platinum-based agent (such as cisplatin), cyclophosphamide,dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine,hydroxyurea, topotecan, irinotecan, azacytidine, vorinostat,ixabepilone, bortezomib, taxanes (e.g., paclitaxel or docetaxel),cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, vinorelbine, colchicin,anthracyclines (e.g., doxorubicin or epirubicin) daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, adriamycin,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, ricin, or maytansinoids.

In one embodiment, the methods of the invention include administering tothe subject an anti-CD47 antibody molecule, e.g., an anti-CD47 antibodymolecule described herein, in combination with an opsonizing antibody.

In embodiments, the opsonizing antibody can facilitate phagocytosis orantibody dependent cellular cytotoxicity (ADCC), or both, of a targetcell, e.g., a tumor cell. In one embodiment, the antigen binding portionof the opsonizing antibody binds to a target antigen, whereas the Fcportion of the opsonizing antibody binds to an Fc receptor on aphagocyte. In other embodiments, the antigen binding portion of theopsonizing antibody binds to a target antigen, whereas the Fc portion ofthe opsonizing antibody binds to an immune effector cell, e.g., via itsFc domain, thus triggering target cell lysis by the bound effector cell(e.g., monocytes, neutrophils and natural killer cells). In oneembodiment, the opsonizing antibody can include one or more of ananti-CD19 antibody, an anti-CD20 antibody, an anti-CD38 antibody, ananti-HER2/neu receptor antibody, an anti-EGFR antibody, an anti-CD30antibody, or an anti-CD33 antibody, either alone or in combination.

The anti-CD47 antibody molecule, e.g., an anti-CD47 antibody moleculedescribed herein, may be administered to the subject in combination witha CD19 inhibitor. The CD19 inhibitor may be an antibody, a fragment orconjugate of an antibody, or a cell therapy. Exemplary anti-CD19antibodies or fragments or conjugates thereof include but are notlimited to blinatumomab, SAR3419 (Sanofi), MEDI-551 (Medlmmune LLC),Combotox, DT2219ARL (Masonic Cancer Center), MOR-208 (also calledXmAb-5574; MorphoSys), XmAb-5871 (Xencor), MDX-1342 (Bristol-MyersSquibb), SGN-CD19A (Seattle Genetics), and AFM11 (Affimed Therapeutics).In certain embodiments, the anti-CD47 antibody molecule may beadministered to the subject in combination with a CD19 inhibitor for thetreatment of cancer, e.g., B-cell lymphomas and leukemias, e.g. acutelymphoblastic leukemia.

The anti-CD47 antibody molecule, e.g., an anti-CD47 antibody moleculedescribed herein, may be administered to the subject in combination witha CD20 inhibitor. The CD20 inhibitor may be a small molecule, anantibody, a fragment or conjugate of an antibody, or a cell therapy.Exemplary anti-CD20 antibodies include but are not limited to rituximab,ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (TrubionPharmaceuticals), ocaratuzumab, and Pro131921 (Genentech). In certainembodiments, an anti-CD47 antibody molecule may be administered to thesubject in combination with a CD20 inhibitor for the treatment of canceror a disorder associated with cancer, e.g., non-Hodgkin's lymphoma,diffuse large B-cell lymphoma, chronic lymphocytic leukemia, NMDAreceptor positive teratoma, or cancer-induced encephalopathy. In certainembodiments, an anti-CD47 antibody molecule may be administered to thesubject in combination with a CD20 inhibitor for the treatment of anautoimmune disease, e.g., rheumatoid arthritis or Myasthenia gravis.

The anti-CD47 antibody molecule, e.g., an anti-CD47 antibody moleculedescribed herein, may be administered to the subject in combination witha CD38 inhibitor. The CD38 inhibitor may be a small molecule, anantibody, a fragment or conjugate of an antibody, or a cell therapy. Oneexemplary anti-CD38 antibody is daratumumab (Johnson & Johnson). Incertain embodiments, the anti-CD47 antibody molecule may be administeredto the subject in combination with a CD38 inhibitor for the treatment ofcancer, e.g. multiple myeloma, B-cell lymphomas, T-cell lymphomas, andleukemias.

The anti-CD47 antibody molecule, e.g., an anti-CD47 antibody moleculedescribed herein, may be administered to the subject in combination witha HER2/neu receptor inhibitor. The anti-HER2/neu receptor inhibitor maybe an antibody, a fragment or conjugate of an antibody, or a celltherapy. One exemplary anti-HER2/neu receptor antibody is trastuzumab(Genentech). In certain embodiments, the anti-CD47 antibody molecule maybe administered to the subject in combination with an anti-HER2/neureceptor antibody for the treatment of cancer, e.g., breast cancer,gastric cancer, e.g., stomach adenocarcinoma, ovarian cancer, lungadenocarcinoma, uterine cancer, salivary duct carcinomas, testiculargerm cell tumors, and esophageal tumors.

Throughout the description, where compositions and kits are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions andkits of the present invention that consist essentially of, or consistof, the recited components, and that there are processes and methodsaccording to the present invention that consist essentially of, orconsist of, the recited processing and method steps.

EXAMPLES

Practice of the invention will be more fully understood from theforegoing examples, which are presented herein for illustrative purposesonly, and should not be construed as limiting the invention in any way.

Example 1—Generation of Anti-CD47 Antibody Molecules

This example describes the production of anti-CD47 antibodies in mice.

Genetically engineered mice carrying a human immunoglobulin immunerepertoire in place of the murine repertoire (Harbour Antibodies BV)were immunized with soluble CD47-Fc fusion protein. Twenty-eighthybridomas expressing anti-CD47 monoclonal antibody molecules wereisolated following fusion of splenocytes with a myeloma cell line,screening and cloning. Isolated hybridomas included hybridomasexpressing antibody molecules referred to as 2.3D11, 4.2B4, 4.2C11,4.1H12, 4.12E2, 2.15A5, 2.7B6, 2.12F6, 2.15E4, 2.3A9, 2.5E6, 2.6D3,4.2C4, 2.3D3, 2.9F9, and 2.1D2. The isolated hybridomas expressedantibody molecules having both heavy and light chains with fully humanvariable domains and rat constant domains.

Exemplary isolated anti-CD47 antibody 2.3D11 (hereafter referred to as“2.3D11”) was sequenced and further characterized below.

The heavy chain of the isolated 2.3D11 antibody has the followingsequence (Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constantregion)

The light chain of the isolated 2.3D11 antibody has the followingsequence (Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constantregion):

Once isolated, the constant regions of the heavy chain were replacedwith heavy chain constant regions from human IgG1 (SEQ ID NO: 17), humanIgG4 (SEQ ID NO: 20) or human IgG4 containing Ser228Pro and Leu235Glusubstitutions (SEQ ID NO: 21), and the constant region of the lightchain was replaced with a human kappa constant region (SEQ ID NO: 22)using conventional recombinant DNA techniques.

The heavy chain of the 2.3D11 antibody comprising a human IgG1 heavychain constant domain (hereinafter referred to as “2.3D11 IgG1”) has thefollowing sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region;without the leader sequence):

The heavy chain of the 2.3D11 antibody comprising a wild-type human IgG4heavy chain constant domain (hereinafter referred to as “2.3D11 IgG4”)has the following sequence (FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constantregion; without leader sequence):

The heavy chain of the 2.3D11 antibody comprising a mutant human IgG4heavy chain constant domain with Ser228Pro and Leu235G1t substitutions(hereinafter referred to as “2.3D11 IgG4mt”) has the following sequence(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region; without the leadersequence, mutated residues boxed):

The light chain of the 2.3D11 antibody comprising a human kappa constantregion has the following sequence(FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Constant region; without the leadersequence):

Example 2: In Vitro Characterization of Anti-CD47 Antibodies

The 2.3D11 antibody generated in Example 1 was tested in a set of invitro assays to ascertain their biological characteristics andactivities. The 2.3D11 antibody was found to potently inhibit theinteraction between CD47 and SIRPα and enhance phagocytosis of tumorcells. Surprisingly and unexpectedly, 2.3D11 was found to cross competewith reference antibody B6H12 for binding to CD47, even though, unlikeB6H12, 2.3D11 does not induce hemagglutination or red blood cellphagocytosis.

I—SIRPα Blocking Activity

SIRPα is a natural ligand of CD47. The ability of 2.3D11 to block theCD47-SIRPα interaction was measured using a flow cytometry based assay,wherein Jurkat cells, which express CD47, were incubated with ananti-CD47 antibody or a control monoclonal antibody (antibodies titrated10 μg/ml-0.17 ng/ml in 3 fold dilution series), washed and thenincubated with SIRPα-Fc-bio (7.5 μg/ml; determined as ˜EC₇₀ fromprevious titration). SIRPα bound to the cells was detected usingstreptavidin-allophycocyanin (SA-APC). As shown in FIG. 1, the 2.3D11antibody potently blocked the CD47-SIRPα interaction.

II—2.3D11 Competes with B6H12 for Binding to CD47

As shown in FIGS. 2A-C, the anti-CD47 antibodies B6H12 and 2.3D11mutually cross compete for (block) binding to CD47, suggesting there isoverlap between the binding epitopes of the two antibodies.

Two approaches were used to study their cross competition for CD47.

First, DU-145 (a human prostate cancer cell line that expresses CD47)cells were pre-incubated with varying concentrations of purifiedanti-CD47 or control antibodies, washed and then stained withbiotinylated versions of the antibodies to assess self- andcross-blocking (FIGS. 2A-B). Biotinylated versions of the antibodieswere detected with streptavidin fluorescein isothiocyanate (SA-FITC).Second, Panc-1 (pancreatic carcinoma cell line that expresses CD47)cells were co-incubated with B6H12 and increasing concentrations ofunlabeled 2.3D11 (FIG. 2C). In both cases, 2.3D11 competed with B6H12for binding to CD47, which indicates that the two antibodies bindoverlapping epitopes.

III—2.3D11 Binding to Cynomolgus Monkey CD47

The ability of 2.3D11 to bind to cynomolgus (cyno) monkey CD47 wasassessed. Briefly, human and cyno red blood cells (RBCs) were isolatedand reacted with a dilution series of each antibody, and analyzed byflow cytometry. As shown in FIG. 3A-D, 2.3D11 binds to both human andcyno RBCs.

IV—2.3D11 Enhances Phagocytosis of Target Cancer Cells

CD47 is a cell surface receptor that is upregulated on tumor cells andis also thought to contribute to immune evasion through its interactionwith its natural ligand SIRPα. Ligation of SIRPα on macrophages by CD47results in decreased phagocytic activity. The effect of the 2.3D11antibody on phagocytosis of target cells was assessed.

Briefly, effector cells (primary human macrophages (CD14+ monocytesisolated from human peripheral blood and differentiated with M-CSF for 7days)), were co-cultured with target cells (carboxyfluoresceinsuccinimidyl ester (CFSE)-labeled Jurkat or Raji cells), at ratiosbetween 1:1 and 1:4 (effector:target), for 2 hours in the presence ofanti-CD47 antibodies or isotype control. Phagocytosis was measured asCD14+CFSE+ events as a percent of the total CD14+ cells as measured byflow cytometry. Cytochalasin D, which inhibits phagocytosis, was used asa control. As shown in FIGS. 4A-B and FIG. 5, the presence of 2.3D11 inco-cultures enhanced phagocytosis of target cells.

Further, as shown in FIG. 6, 2.3D11 cooperates with the anti-CD20antibody rituximab to promote Raji cell phagocytosis. These resultssuggest that tumor cell phagocytosis can be enhanced in the presence ofopsonizing antibodies (e.g., anti-CD20 antibodies) when co-administeredwith 2.3D11. Additionally, as shown in FIGS. 7A-C, phagocytosis may beinfluenced by the level of CD47 expression on target cell. 2.3D11increased phagocytosis of Raji tumor cell line targets with an EC₅₀ of˜300 ng/mL (data not shown).

V—Hemagglutination Activity of 2.3D11

To evaluate the hemagglutinating capacity of 2.3D11, human RBCs wereincubated with a dose range of anti-CD47 antibody, for example, 2.3D11,4.2B4, 4.2C11, 4.1H12, 4.12E2, 2D3, B6H12, and AB6.12-IgG4PE, or controlin a 96 well plate. Evidence of hemagglutination was demonstrated by thepresence of non-settled RBCs, appearing as a haze compared to a punctatedot of non-hemagglutinated RBCs.

Unexpectedly, as shown in FIG. 8, antibody 2.3D11 did not exhibithemagglutinating activity at any of the concentrations tested despitebinding competition with B6H12 antibody, which is known to causehemagglutination.

VI—2.3D11 does not Enhance Phagocytosis of Target Red Blood Cells

To evaluate whether binding of 2.3D11 to RBC leads to increasedphagocytic uptake by macrophages, phagocytosis assays similar to thosedescribed in section IV above were performed, using human or cyno RBC astargets at an effector:target ratio of 1:10. As shown in FIG. 9, 2.3D11had minimal effect on human and cyno RBC phagocytosis, in contrast toB6H12 which enhanced phagocytosis.

In summary, increased phagocytosis mediated by 2.3D11 is preferentialfor tumor cells over normal leukocytes and RBC.

Example 3—In Vivo Efficacy of Anti-CD47 Antibody in Tumor Models

The anti-tumor activity of 2.3D11, produced as either a wild-type humanIgG4 (“2.3D11 IgG4”) or S228P/L235E double mutant human IgG4 (“2.3D11IgG4mt”), as described in Example 1, was evaluated in the Burkitt'slymphoma Raji xenograft model.

Female CB.17 SCID mice were injected subcutaneously with 1×10⁷ Raji Btumor cells in 50% Matrigel and treatment was started when tumorsreached 100 mm³. Isotype control, 2.3D11 IgG4 and 2.3D11 IgG4mtantibodies were injected intraperitoneally (i.p.) three times per week,for 3 weeks at the indicated doses. Rituximab was injected i.p. at 5mg/kg once a week for 3 weeks. Body weight and tumor volume weremeasured twice per week.

The antitumor efficacy of 2.3D11 IgG4 and 2.3D11 IgG4mt (200 μg/mouse,t.i.w.) were compared in the Raji model of Burkitt's lymphoma. As shownin FIG. 10A, both the 2.3D11 IgG4 and 2.3D11 IgG4mt antibodiesdemonstrated anti-tumor activity in this xenograft model. At the timethe isotype control group reached 2000 mm³ (day 24), tumor growthinhibition (TGI) activity of the 2.3D11 IgG4 and 2.3D11 IgG4mtantibodies was 97% and 71%, respectively.

In the Raji xenograft model, the anti-tumor activity of 2.3D11derivatives was at least partially dependent on macrophages, asdepletion of macrophages via clodronate administration led to reducedtumor growth inhibition. Tumor-associated macrophage (TAM) numbers andpolarization status were also modulated by 2.3D11 derivative treatment(data not shown).

The Raji model has been shown to be sensitive to rituximab, an anti-CD20antibody used as a first line therapy for diffuse large B-cell lymphomapatients. The antitumor efficacy of 2.3D11 IgG4 (100 μg/mouse, t.i.w.)and 2.3D11 IgG4mt (200 μg/mouse, t.i.w.) in combination with rituximab(5 mg/kg, q.w.) was assessed in the Raji model. The results for 2.3D11IgG4mt at day 19 after the start of treatment are summarized in FIG.10B. TGI activity of 2.3D11 IgG4mt antibody alone was 51% and rituximabTGI was 67%. When combined, 2.3D11 IgG4mt and rituximab achieved 96%TGI, indicating synergistic improvement of tumor growth inhibition bythe combined antibodies. FIG. 10C summarizes the results for 2.3D11 IgG4at day 19 after the start of treatment. The data show that 2.3D11 IgG4is highly potent in a monotherapy setting, leading to tumor regression(from 124 mm³ at the start of treatment to 47 mm³ at day 19) and 96%TGI, similar to the results in the experiment described above but usingonly half the amount of 2.3D11 IgG4 antibody. The high potency of the2.3D11 IgG4 makes it difficult to assess the possible additional effectof the combination with rituximab. However, it is noteworthy that at anearlier time point, day 12, the 2.3D11 IgG4 arm had only 1 tumor-freemouse, whereas the combination arm had 5 tumor free mice. In all theexperiments described above, no body weight loss was reported.

In summary, 2.3D11 administration led to profound tumor growthinhibition in a model of Burkitt's lymphoma as a single agent and incombination with an opsonizing antibody.

Example 4—Fc Format Variants of Anti-CD47 Antibody

The activity of 2.3D11, produced in three different Fc formats, wasevaluated in multiple assays. 2.3D11 was produced with wild-type humanIgG4 (“2.3D11 IgG4”) or S228P/L235E double mutant human IgG4 (“2.3D11IgG4mt”) or wild type IgG1 (“2.3D11 IgG1”).

I—RBC Phagocytosis

Human red blood cells (RBCs) were isolated from healthy donors andlabeled with CFSE. Labeled RBCs were cultured with day 7 humanmacrophages in the presence of a 2.3D11 antibody, isotype control, oranti-CD47 antibody B6H12 for two hours at a target-to-effector ratio of10:1. After culture, cells were trypsinized and stained withanti-CD14-APC and analyzed by flow cytometry.

Phagocytosis was quantitated as the percent of CD14+ events(macrophages) that were also CFSE+ and had therefore engulfed a target(events were gated on singlets). No significant difference was observedbetween the isotype controls or the 2.3D11 IgG1, 2.3D11IgG4 or2.3D11IgG4mt antibodies, but B6H12 strongly induced RBC phagocytosis.Representative data is shown in FIG. 11.

II—Phagocytosis by Polarized Macrophages

Primary human monocytes were differentiated in 100 ng/mL recombinanthuman macrophage colony-stimulating factor (M-CSF) for 6 days. On thesixth day, macrophages were replated in the presence of either (A) M-CSFalone, producing unpolarized macrophage, (B) M-CSF plus interleukin-10(IL-10), transforming growth factor β (TGFβ) and interleukin-4 (IL-4),polarizing the macrophage to the M2 phenotype, (C) M-CSF plus interferonγ and lipopolysaccharide (LPS), polarizing the macrophage to the M1phenotype, or (D) M-CSF plus dexamethasone (Dex), polarizing themacrophage to a strong M2 phenotype, overnight.

Phagocytosis assays were performed on day 7, as described above, usingCFSE-labeled Jurkat cells as targets. The results are summarized inFIGS. 12A-12D, which demonstrate that the anti-CD47 antibody 2.3D11,regardless of Fc format, enhances phagocytosis by both M1 and M2polarized macrophages.

III—Tumor Cell Phagocytosis

Primary human monocytes were differentiated in 100 ng/mL recombinanthuman macrophage colony-stimulating factor (M-CSF) for 7 days. Frozenbone marrow samples from AML patients or healthy donors were thawed,labelled with CFSE and cultured with differentiated macrophages for 2hours at a target-to-effector ratio of 1:1, in the presence of either 10or 5 μg/mL of the indicated antibodies. Phagocytosis was quantitated asdescribed above. The results are summarized in FIG. 13, whichdemonstrate that both the 2.3D11 IgG1 and 2.3D11 IgG4 anti-CD47antibodies stimulate phagocytosis of bone marrow cells from AMLpatients.

IV—Burkitt's Lymphoma Raji Xenograft Model

SCID-Beige mice were injected subcutaneously with 1×10⁷ Raji B tumorcells in 50% Matrigel and treatment was started when tumor reached 100mm³. Isotype control (polyclonal human IgG), 2.3D11 IgG4, 2.3D11 IgG4mtand 2.3D11IgG1 antibodies were injected intraperitoneally (i.p.) with200 μg of antibody three times per week for 3 weeks. Body weight andtumor volume were measured twice per week.

As shown in FIG. 14, the 2.3D11 IgG1, 2.3D11 IgG4 and 2.3D11 IgG4mtanti-CD47 antibodies demonstrated anti-tumor activity in this xenograftmodel, but the 2.3D11 IgG4mt antibody showed significantly less tumorgrowth inhibition than either the 2.3D11 IgG4 or 2.3D11 IgG1 antibodies.

Example 5—Antibody 2.3D11 and Anti-CD38 Antibody Act Synergistically toEnhance Macrophage Phagocytosis of Multiple Myeloma Cells

This example shows that a 2.3D11 derived antibody acts synergisticallywith an anti-CD38 opsonizing antibody.

Primary human monocytes were differentiated in 100 ng/mL recombinanthuman macrophage colony-stimulating factor (M-CSF) for 7 days. A primarymultiple myeloma bone marrow sample was CFSE labeled and co-culturedwith differentiated human macrophages at a ratio of 2:1, in the presenceof 10 μg/mL of antibody 2.3D11 IgG4, an anti-human CD38-hIgG1 antibody(MAB1135, G&P Biosciences), or both (single-agent conditions weresupplemented with 10 μg/mL of isotype control).

Phagocytosis was assessed by flow cytometry and reported as percent ofmacrophages that are CFSE-positive. The results are summarized in FIG.15, which shows that the combination of an anti-CD47 antibody with ananti-CD38 antibody synergistically enhances phagocytosis of multiplemyeloma cells as compared to either antibody alone.

In summary, the results presented herein demonstrate that the anti-CD47antibody 2.3D11 induces robust tumor cell phagocytosis and tumorclearance both alone and in combination with opsonizing antibodies inpreclinical models of multiple myeloma.

Example 6—Antibody 2.3D11 and an Anti-CD38 Antibody Act Synergisticallyto Reduce Tumor Burden in Murine Xenograft Model of Multiple Myeloma

This example describes the synergistic properties that can be observedwhen a 2.3D11 derived antibody is combined with an anti-CD38 opsonizingantibody in a murine model of multiple myeloma.

8-12 week old CB.17 SCID female mice (Charles River) were injectedsubcutaneously with 1×10⁷ H929 tumor cells in 50% Matrigel in the rightflank. Cell injection volume was 0.1 mL/mouse. When tumors reached anaverage size of 100-150 mm³, animals were randomized to control ortreatment. Treatment groups included 2.3D11 IgG4 at 30 μg/mouse(injected intraperitoneally (i.p.) three times per week for 3 weeks),daratumumab at 10 μg/mouse (injected i.p. at a single dose), and acombination of the two antibodies. Tumor volumes were measured twiceweekly with a caliper using the formula (length*width2*0.52). Theresults are show in FIG. 16 and demonstrate that anti-tumor activity ofthe combination of 2.3D11 IgG4 and daratumumab is greater than eithersingle agent alone.

Example 7—Antibody 2.3D11 Enhances Phagocytosis of Chronic LymphocyticLeukemia (CLL) Cells

This example describes enhanced phagocytosis of chronic lymphocyticleukemia (CLL) cells mediated by a 2.3D11 derived antibody.

Primary human monocytes were differentiated in 100 ng/mL recombinanthuman macrophage colony-stimulating factor (M-CSF) for 7 days.CD19+/CD5+ tumor cells from the peripheral blood of a CLL patient wereCFSE labeled and co-cultured with differentiated human macrophages at aratio of 2:1 for two hours, in the presence of antibody 2.3D11 IgG4, orisotype control (anti-DNP antibody with a hIgG4 constant region).Phagocytosis was assessed by flow cytometry, as described above, andreported as percent of CD14+macrophages that are CFSE-positive.

The results are summarized in FIG. 17, which demonstrate the ability of2.3D11 IgG4 to significantly boost the phagocytosis of primary CLL cellsby macrophages in vitro. These data suggest that different stages of CLLmay respond to 2.3D11 IgG4 treatment.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles cited herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. An isolated monoclonal antibody that specificallybinds human CD47, or antigen binding fragment thereof, wherein themonoclonal antibody or antigen binding fragment thereof comprises: aheavy chain complementarity determining region 1 (HC CDR1) comprisingthe amino acid sequence set forth in SEQ ID NO: 7; a heavy chaincomplementarity determining region 2 (HC CDR2) comprising the amino acidsequence set forth in SEQ ID NO: 8; a heavy chain complementaritydetermining region 3 (HC CDR3) comprising the amino acid sequence setforth in SEQ ID NO: 9; a light chain complementarity determining region1 (LC CDR1) comprising the amino acid sequence set forth in SEQ ID NO:10; a light chain complementarity determining region 2 (LC CDR2)comprising the amino acid sequence set forth in SEQ ID NO: 11; and alight chain complementarity determining region 3 (LC CDR3) comprisingthe amino acid sequence set forth in SEQ ID NO:
 12. 2. The isolatedmonoclonal antibody or antigen-binding fragment of claim 1, wherein theantibody or antigen-binding fragment inhibits the interaction betweenhuman CD47 and SIRPα.
 3. The isolated monoclonal antibody orantigen-binding fragment of claim 1, wherein the antibody orantigen-binding fragment does not cause significant, or detectable,hemagglutination of human erythrocytes relative to a referencemonoclonal antibody that specifically binds human CD47 and causessignificant, or detectable, hemagglutination of human erythrocytes, orantigen binding fragment thereof.
 4. The isolated monoclonal antibody orantigen-binding fragment of claim 1, wherein the antibody is a humanantibody.
 5. The isolated monoclonal antibody or antigen-bindingfragment of claim 1, wherein the antibody comprises a wild type humanIgG1 or wild type human IgG4 heavy chain constant region.
 6. Theisolated monoclonal antibody or antigen-binding fragment of claim 1,wherein the antibody comprises a mutant IgG1 heavy chain constantregion.
 7. The isolated monoclonal antibody or antigen-binding fragmentof claim 6, wherein the mutant IgG1 heavy chain constant regioncomprises a substitution at Glu233, Leu234, Leu235, Asn297, or acombination thereof, numbering according to EU numbering.
 8. Theisolated monoclonal antibody or antigen-binding fragment of claim 6,wherein the mutant IgG1 heavy chain constant region comprises an E233Psubstitution, an L234A or L234E substitution, an L235A substitution, anN297A substitution, or a combination thereof, numbering according to EUnumbering.
 9. The isolated monoclonal antibody or antigen-bindingfragment of claim 1, wherein the antibody comprises a wild type humanIgG4 heavy chain constant region.
 10. The isolated monoclonal antibodyor antigen-binding fragment of claim 1, wherein the antibody comprises amutant IgG4 heavy chain constant region.
 11. The isolated monoclonalantibody or antigen-binding fragment of claim 10, wherein the mutantIgG4 heavy chain constant region comprises a substitution at Ser228,Leu235, Asn297, or a combination thereof, numbering according to EUnumbering.
 12. The isolated monoclonal antibody or antigen-bindingfragment of claim 10, wherein the mutant IgG4 heavy chain constantregion comprises an S228P substitution, an L235E substitution, an N297Asubstitution, or a combination thereof, numbering according to EUnumbering.
 13. The isolated monoclonal antibody or antigen-bindingfragment of claim 10, wherein the mutant IgG4 heavy chain constantregion comprises an S228P substitution and an L235E substitution,numbering according to EU numbering.
 14. The isolated monoclonalantibody or antigen-binding fragment of claim 5, wherein the antibodycomprises a human kappa constant region.
 15. An isolated monoclonalantibody that specifically binds human CD47 or an antigen-bindingfragment thereof, wherein the monoclonal antibody comprises a heavychain variable region (VH) comprising the amino acid sequence set forthin SEQ ID NO: 4 and a light chain variable region (VL) comprising theamino acid sequence set forth in SEQ ID NO:
 6. 16. The isolatedmonoclonal antibody or antigen-binding fragment of claim 15, wherein theantibody or antigen-binding fragment inhibits the interaction betweenhuman CD47 and SIRPα.
 17. The isolated monoclonal antibody orantigen-binding fragment of claim 15, wherein the antibody orantigen-binding fragment does not cause significant, or detectable,hemagglutination of human erythrocytes relative to a referencemonoclonal antibody that specifically binds human CD47 and causessignificant, or detectable, hemagglutination of human erythrocytes, orantigen binding fragment thereof.
 18. The isolated monoclonal antibodyor antigen-binding fragment of claim 15, wherein the antibody is a humanantibody.
 19. The isolated monoclonal antibody or antigen-bindingfragment of claim 15, wherein the antibody comprises a wild-type humanIgG1 or wild-type human IgG4 heavy chain constant region.
 20. Theisolated monoclonal antibody or antigen-binding fragment of claim 19,wherein the antibody comprises a human kappa constant region.
 21. Theisolated monoclonal antibody or antigen-binding fragment of claim 19,wherein the wild-type human IgG4 heavy chain constant region comprisesthe amino acid sequence set forth in SEQ ID NO:
 20. 22. The isolatedmonoclonal antibody or antigen-binding fragment of claim 20, wherein thehuman kappa constant region comprises the amino acid sequence set forthin SEQ ID NO:
 22. 23. An isolated monoclonal antibody that specificallybinds human CD47, wherein the monoclonal antibody comprises: a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 15, SEQID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25; and a light chain comprisingthe amino acid sequence set forth in SEQ ID NO: 16 or SEQ ID NO:
 26. 24.The isolated monoclonal antibody of claim 23, wherein the antibodyinhibits the interaction between human CD47 and SIRPα.
 25. The isolatedmonoclonal antibody of claim 23, wherein the antibody does not causesignificant, or detectable, hemagglutination of human erythrocytesrelative to a reference monoclonal antibody that specifically bindshuman CD47 and causes significant, or detectable, hemagglutination ofhuman erythrocytes, or antigen binding fragment thereof.
 26. Theisolated monoclonal antibody of claim 23, wherein the antibody is ahuman antibody.
 27. An isolated monoclonal antibody that specificallybinds human CD47, wherein the monoclonal antibody comprises: a heavychain comprising the amino acid sequence set forth in SEQ ID NO: 24; anda light chain comprising the amino acid sequence set forth in SEQ ID NO:26.
 28. The isolated monoclonal antibody of claim 27, wherein theantibody inhibits the interaction between human CD47 and SIRPα.
 29. Theisolated monoclonal antibody of claim 27, wherein the antibody does notcause significant, or detectable, hemagglutination of human erythrocytesrelative to a reference monoclonal antibody that specifically bindshuman CD47 and causes significant, or detectable, hemagglutination ofhuman erythrocytes, or antigen binding fragment thereof.
 30. Acomposition comprising the isolated monoclonal antibody orantigen-binding fragment of claim 1 and a pharmaceutically acceptablecarrier or diluent.